MEDICAL    SCHOOL 
LIIB1RAIKV 


Gift  of 
Glanvilie  Y.   Rusk,   M.D. 


A  TEXT-BOOK 


OF 


ACTERIOLOGY 


BY 

GEORGE  M.  STERNBERG,  M.D.,  LL.D. 

SURGEON- GENERAL  U.S.    ARMY 

EX-PRESIDENT  AMERICAN  PUBLIC    HEALTH    ASSOCIATION;   HONORARY  MEMBER  OP  THE  EPIDEMIOLOGICAL 

SOCIETY  OP  LONDON,   OP  THE   ROYAL  ACADEMY  OP  MEDICINE  OP  ROME,   OP  THE  ACADEMY 

OP  MEDICINE  OF  RIO  DE  JANEIRO,   OP  THE  SOCIETE  FRANCAISE  D'HYGIENE, 

ETC.,   ETC. 


ILLUSTRATED  BY  HELIOTYPE  AND  CHROMO-LITHOGRAPHIC  PLATES 
AND  TWO  HUNDRED  ENGRAVINGS. 


NEW  YORK 
WILLIAM    WOOD   AND   COMPANY 

1896 


COPYRIGHT  BY 

.1.1  AM      WOOD     &     COMPANY, 
189C. 


PREFACE. 


THE  writer's  Manual  of  Bacteriology,  published  in  1892,  has  been 
very  favorably  received  both  in  this  country  and  abroad,  but  its  use- 
fulness has  no  doubt  been  to  some  extent  restricted  by  the  size  and 
expense  of  the  volume.  The  following  is  an  extract  from  the  preface 
of  the  Manual : 

"  A  Manual  of  Bacteriology,  therefore,  which  fairly  represents  the 
present  state  of  knowledge,  will  consist  largely  of  a  statement  of  facts 
established  by  experimental  data,  and  cannot  fail  to  be  of  value  to 
physicians  and  to  advanced  students  of  bacteriology  as  a  work  of 
reference.  The  present  volume  is  an  attempt  to  supply  such  a  man- 
ual, and  at  the  same  time  a  text-book  of  bacteriology  for  students 
and  guide  for  laboratory  work.  That  portion  of  the  book  which  is 
printed  in  large  type  will,  it  is  hoped,  be  found  to  give  an  accurate 
and  sufficiently  extended  account  of  the  most  important  pathogenic 
bacteria,  and  of  bacteriological  technology,  to  serve  as  a  text-book  for 
medical  students  and  others  interested  in  this  department  of  science. 
The  descriptions  of  non-pathogenic  bacteria,  and  of  the  less  important 
or  imperfectly  described  species  of  pathogenic  bacteria,  are  given  in 
smaller  type." 

For  the  benefit  of  students  of  medicine  and  others  who  do  not  care 
especially  for  the  detailed  descriptions  of  non-pathogenic  bacteria  and 
the  extensive  bibliography  contained  in  the  Manual,  this  TEXT-BOOK 
OF  BACTERIOLOGY  is  now  published.  It  comprises  that  portion  of  the 
Manual  above  referred  to  as  printed  in  large  type,  revised  to  include 
all  important  additions  to  our  knowledge  of  the  pathogenic  bacteria 
since  the  original  date  of  publication. 


TABLE   OF   COISTTEOTS. 


PART  FIRST. 

CLASSIFICATION,  MORPHOLOGY,  AND  GENERAL  BACTERIOLOGICAL 

TECHNOLOGY. 

PAGE 

I.  HISTORICAL, 3 

II.  CLASSIFICATION, 10 

III.  MORPHOLOGY, 20 

IV.  STAINING  METHODS, 25 

V.  CULTURE  MEDIA 37 

VI.  STERILIZATION  OF  CULTURE  MEDIA, 50 

VII.  CULTURES  IN  LIQUID  MEDIA, 60 

VIII.  CULTURES  IN  SOLID  MEDIA, 67 

IX.  CULTIVATION  OF  ANAEROBIC  BACTERIA, 78 

X.  INCUBATING  OVENS  AND  THERMO-REGULATORS,          .  .86 

XI.  EXPERIMENTS  UPON  ANIMALS, 94 

XII.  PHOTOGRAPHING  BACTERIA, 101 


PART   SECOND. 
GENERAL  BIOLOGICAL  CHARACTERS. 

I.  STRUCTURE,  MOTIONS,  REPRODUCTION, 115 

II.  CONDITIONS  OF  GROWTH, 122 

III.  MODIFICATIONS  OF  BIOLOGICAL  CHARACTERS,    .        .        .        .126 

IV.  PRODUCTS  OF  VITAL  ACTIVITY, 130 

V.  PTOMAINES  AND  Tox ALBUMINS,     ....  .        .  143 

VI.  INFLUENCE  OF  PHYSICAL  AGENTS, .149 

VII.  ANTISEPTICS  AND  DISINFECTANTS  (GENERAL  ACCOUNT  OF  THE 

ACTION  OF), 160 

VIII.  ACTION  OF  GASES  AND  OF  THE  HALOID  ELEMENTS  UPON  BAC- 
TERIA,            .168 

IX.  ACTION  OF  ACIDS  AND  ALKALIES,       ....  .176 

X.  ACTION  OF  VARIOUS  SALTS, 

XI.  ACTION  OF  COAL-TAR  PRODUCTS,  ESSENTIAL  OILS,  ETC.,          .  193 

XII.  ACTION  OF  BLOOD  SERUM  AND  OTHER  ORGANIC  LIQUIDS,        .  204 

XIII.  PRACTICAL  DIRECTIONS  FOR  DISINFECTION,         .        .        .        .208 


ri  TABLE   OP    CONTENTS. 

PART    THIRD. 
PATHOGENIC  BACTERIA. 

PAGE 

I.  MODES  OF  ACTION, 221 

1 1 .  CHANNELS  OP  INFECTION 229 

III.  SUSCEPTIBILITY  AND  IMMUNITY, 233 

IV.  I'VOOENIC  BACTERIA 275 

V.  I:\.-TKIM  \  IN  CROUPOUS  PNEUMONIA, 300 

VI.  PATHOGENIC   MIOROCOCCI  NOT   DESCRIBED   IN   SECTIONS    IV. 

AND  V., 322 

VII.  THE  BACILLUS  OF  ANTHRAX, 339 

VMI.  THE  BACILLUS  OF  TYPHOID  FEVER,     ....  .349 

IX  r.v<  iKitiA  IN  DIPHTHERIA, 371 

X.  BACTERIA  IN  INFLUENZA 387 

X  I .  BACILLI  IN  CHRONIC  INFECTIOUS  DISEASES 392 

XII.  BACILLI   WHICH  PRODUCE   SEPTICAEMIA  IN  SUSCEPTIBLE   ANI- 
MALS  428 

X 1 1  [.  PATHOGENIC  AEROBIC  BACILLI  NOT  DESCRIBED  IN   PREVIOUS 

SECTIONS, 461 

XIV.  PATHOGENIC  ANAEROBIC  BACILLI,        .  ...  531 

XV.  PATHOGENIC  SPIRILLA,    ....  ....  549 

XVI.  BACTERIA  IN  INFECTIOUS  DISEASES,  ....  571 


PART  FOURTH. 
SAPROPHYTES. 

I.  BACTERIA  IN  THE  AIR 623 

1 1    BACTERIA  IN  WATER, 636 

III    i'.ACTERIA  IN  THE  SOIL, 652 

IV.  BACTERIA  OF  THE  SURFACE  OF  THE  BODY  AND  OF  EXPOSED 

Mucous  MEMBRANES 658 

v    BACTERIA  OF  THE  STOMACH  AND  INTESTINES,    ....  668 
vi    I:\.TKIM.V  OF  CADAVERS  AND  OF  PUTREFYING  MATERIAL  FROM 

VARIOUS  SOURCES 674 

vil.  BACTERIA  IN  ARTICLES  OF  FOOD, 677 

INDEX, .683 


LIST  OF  ILLUSTRATIONS. 


PAGE 

1.  Staphylococci,           .        .        .        .        .        .                         .        .        .  21 

2.  Zoogloea 21 

3.  Ascococcus, 21 

4.  Streptococci, 21 

5  Tetrads,    .        . 22 

6.  Packets— sarcina, .        .        .  22 

7.  Bacilli, 23 

8.  Involution  forms,      ...........  23 

9.  Chains  formed  by  binary  division,    ........  23 

10.  Spirilla, 24 

11.  Cladothrix 24 

12.  Flagella, 24 

13.  Platinum  wire  in  glass  handle,          ........  25 

14.  Flask  for  drawing  off  blood  serum, .  38 

15.  Method  of  forcing  blood  serum  into  test  tube, 38 

16.  Suction  pipette, 38 

17.  Hot- water  funnel, 42 

18.  Karlinski's  agar  filter,       .        .        .        . 44 

19.  Unna's  agar  filter, 45 

20.  Glass  dishes  for  preserving  potato  cultures, 48 

21.  Test  tube  for  sterilizing  potato,        .                 48 

22.  Shape  of  potato  for  test-tube  culture, 48 

23.  Hot  air  oven,    . 52 

24.  Koch's  steam  sterilizer, .  53 

25.  Koch's  steam  sterilizer, 53 

26.  Arnold's  steam  sterilizer,           ...                 54 

27.  Miincke's  steam  sterilizer,         .........  54 

28.  Koch's  apparatus  for  coagulating  blood  serum,      .....  55 

29.  Miincke  s  steam  sterilizer  and  coagulator, 56 

30.  Pasteur  Chamberlain  filter, 57 

31.  Pasteur- Chamberlain  filter  without  metal  case 58 

32.  Modified  Pasteur-Chamberlain  filter, 59 

33.  Erlenmeyer  flask 6* 

34.  Flask  used  by  Pasteur, 61 

35.  Platinum  wire  loop, 62 

36.  Platinum  needle, 63 

37.  Sternberg's  bulb, • 

38.  Fermentation  tube, 66 

39.  Method  of  making  stick  culture, 6? 


iji  LIST  OF  ILLUSTRATIONS. 

PAGE 

o.  68 

40.  Sloping  surface  of  culture  medium, 

40A.  Growth  of  non-liquefying  bacteria  in  gelatin  stick  cultures,  . 

41.  Growth  of  same  along  line  of  puncture,  ...  ^ 

42.  Growth  of  liquefying  bacilli,   ...  ^ 

43.  Colonies  of  bacteria,  73 

44.  Apparatus  for  gelatin  plates,    .        .  ^ 
Ksmarch  roll  tube 

46.    (See  Fig.  15).  7S 

47     Mode  of  development  of  a  facultative  anaerobic  bacillus,       . 

48.  Mode  of  development  of  strict  anaerobic  in  long  stick  culture,       .        .          78 

49.  Exhausted-air  flask  for  liquid  media, 

50.  Method  of  displacing  air  with  hydrogen,  ™ 

51.  Salomonson's  tube 

52.  Frftnkel's  method  of  cultivation 

58.  Sternberg's  method  of  cultivation 

54.  Sternberg's  method  of  cultivation 

55.  Buchner's  method  of  cultivation, 

56.  Hydrogen  generator, 

Hydrogen  apparatus  for  plate  cultures  . 

58.  Incubating  oven 

59.  Thermo-regulator  for  gas 

60.  Moitessier's  pressure  regulator, 

r,i.  Mica  screen  for  flame, 

62.  Koch's  device  for  cutting  off  flame 

63.  Heichert's  thermo  regulator, 

64.  Bohr's  thermo-regulator 

65.  MQncke's  thermo- regulator 

66.  Sternberg's  thermo-regulator 

67.  Gas  valve  for  the  same 

68.  D'Arsonval's  incubating  apparatus, 

69.  Roux's  incubating  oven  and  thermo-regulator, 

70.  Koux's  thermo-regulator 

71.  Koch's  syringe 

7J.  Sternberg's  glass  syringe 

78.  Pringle's  photomicrographic  apparatus,  . 

74.  Sternberg's  photomicrographic  apparatus  for  gas, 

75.  Spores  of  bacilli 

76.  Method  of  germination  of  spores, 

77.  Apparatus  for  cultivating  anaerobic  bacilli l^-> 

Bacillus  of  mouse  septicaemia  in  leucocytes  from  blood  of  mouse,          .         ~~><> 

79.  Staphylococcus  pyogenes  aureus, 

80.  Gelatin  culture  of  Staphylococcus  pyogenes  aureus,       .        .  .        27 :» 

81.  Vertical  section  through  a  subcutaneous  abscess  caused  by  inoculation 

with  staphylococd  in  the  rabbit 281 

88.    Pus  containing  streptococci, 2*7 

88.    Streptococcus  of  erysipelas  in  nutrient  gelatin, 288 

84.  Section  from  margin  of  un  erysipelatous  inflammation,  showing  strepto- 

cocci in  lymph  spaces, 289 

85.  Gouococci 2J»."> 

86.  Gonococcus  in  gonorrhoea!  pus 2JW 

87.  Gonorrhoea  1  conjunctivitis,  second  day  of  sickness,         .        .  .  298 


LIST    OF   ILLUSTRATIONS.  ix 

FIG.  PAGE 

88.  Friedlander's  bacillus, 308 

89.  Friedlander's  bacillus;  stick  culture  in  gelatin, 309 

90.  Micrococcus  pneumonise  crouposae, 313 

91.  Micrococcus  pneumonias  crouposae, 313 

92.  Micrococcus  pneumoniae  crouposae, 313 

93.  Micrococcus  pneumoniae  crouposae,  showing  capsule,      .        .        .  314 

94.  Single  colony  of  Micrococcus  pneumoniae  crouposae  upon  agar  plate,     .  315 

95.  Micrococcus  pneumoniae  crouposae  in  blood  of  rabbit  inoculated  with 

sputum, 319 

96.  Microcoecus  of  progressive  tissue  necrosis  in  mice,          ....  328 

97.  Micrococcus  of  pyaemia  in  rabbits,   .* 334 

98.  Micrococcus  tetragenus, 326 

99.  Streptococcus  of  mastitis  in  cows, 333 

100.  Bacillus  anthracis,  showing  development  of  long  threads  in  convoluted 

bundles, 340 

101.  Bacillus  anthracis,  showing  formation  of  spores, 341 

102.  Culture  of  Bacillus  anthracis  in  nutrient  gelatin,     .        .         .         .         .  342 

103.  Colonies  of  Bacillus  anthracis  upon  gelatin  plates,          ....  343 

104.  Bacillus  anthracis  in  liver  of  mouse 346 

105.  Bacillus  anthracis  in  kidney  of  rabbit, 347 

106.  Bacillus  of  typhoid  fever;  colonies  in  stained  sections  of  spleen,     .        .  353 

107.  Bacillus  of  typhoid  fever;  colonies  in  stained  sections  of  spleen,     .        .  352 

108.  Bacillus  typhi  abdominalis, 358 

109.  Bacillus  typhi  abdominalis, 358 

110.  Bacillus  typhi  abdominalis,  showing  flagella, 359 

111.  Single  colony  of  Bacillus  typhi  abdominalis  in  nutrient  gelatin,     .        .  360 

112.  Bacillus  typhi  abdomiualis;  stick  culture  in  nutrient  gelatin,          .        .  360 

113.  Section  through  wall  of  intestine,  showing  invasion  by  typhoid  bacilli,  364 

114.  Bacillus  diphtheriae, 375 

115.  Colonies  of  Bacillus  diphtheriae  in  nutrient  agar,    .....  376 

116.  Bacillus  tuberculosis,        ..........  394 

117.  Bacillus  tuberculosis  in  sputum,       ........  395 

lib.  Section  through  a  tuberculous  nodule  in  the  lung  of  a  cow,  showing 

two  giant  cells, 397 

119.  Tubercle  bacilli  from  surface  of  culture  upon  blood  serum,    .        .        .  400 

120.  Culture  of  tubercle  bacillus  upon  glycerin-agar, 402 

121.  Limited  epithelioid- celled  tubercle  of  the  iris, 408 

122.  Section  of  a  recent  lepra  nodule  of  the  skin, 414 

123.  Bacillus  mallei, 417 

124.  Section  of  a  glanders  nodule,  .........  417 

125.  Section  through  a  glanders  nodule  in  liver  of  field  mouse,       .        .        .  420 

126.  Migrating  cell  containing  syphilis  bacilli,        ......  423 

127.  Pus  from  hard  chancre  containing  syphilis  bacilli,           423 

128.  Bacillus  of  rhinoscleroma  in  lymphatic  vessels  of  the  superficial  part  of 

tumor,  .............  425 

129.  Bacillus  septicaemiae  haemorrhagicae  in  blood  of  a  rabbit,        .        .        .  439 

130.  Bacillus  septicaemiae  haemorrhagicae;  stick  culture  in  nutrient  gelatin,  .  431 

131.  Bacillus  of  Schweineseuche, 431 

132.  Colonies  of  bacillus  of  swine  plague,        .......  431 

133.  Bacillus  of  Schweineseuche  in  blood  of  rabbit, 433 

134.  Bacillus  of  hog  cholera,   ...                 ...                .  435 


OF   ILLUSTRATION-. 


PAGE 


185.  Bacillus  of  mouse  septictemia  in  leucocytes  from  blood  of  mouse, 

186.  Bacillus  of  rouget, • 

Ilacillus  of  mouse  septicaemia  ;  culture  in  nutrient  gelatin,     . 

188.     Bacillus  of  mouse  septicaemia;  single  colony  in  nutrient  gelatin,     . 
m    Section  of  diaphragm  of  a  mouse  dead  from  mouse  septicaemia,     . 

140.  Bacillus  cavicida  Havaniensis, 

141.  Bacillus  crassus  sputigenus ™ 

Proteushominiscapsulatus, *5* 

148.    Bacillus  capsulatus, 

144.  Bacillus  hydrophilus  fuscus, ... 

145.  Culture  of  Bacillus  hydrophilus  fuscus  in  nutrient  gelatin,     . 

146.  Bacillus  coli  communis, 

1 5:u  illus  coli  communis  in  nutrient  gelatin, 46* 

148.  A  port  ion  of  the  growth  shown  in  Fig.  147, 465 

149.  Bacillus  lactis  aSrogenes, 472 

150.  Bacillus  acidiformuns 4^4 

151.  Culture  of  Bacillus  acidiformans  in  nutrient  gelatin,      ....  4^4 

152.  Bacillus  cuniculicida  Havaniensis 4^5 

158.    Colonies  of  Bacillus  cuniculicida  Havaniensis, 476 

154.  Colonies  of  Bacillus  cuniculicida  Havaniensis 476 

155.  Bacillus  pyocyanus, 479 

156.  Proteus  vulgaris 485 

157.  "  Swarming  islands"  from  a  culture  of  Proteus  mirabilis,     .        .        .  490 

158.  Spiral  zooglcea  from  a  culture  of  Proteus  mirabilis,        ....  490 

159.  Bacillus  gradlis  cadaveris 516 

160.  Colonies  of  B;u  ill  us  gracilis  cadaveris,  . 516 

161.  Tetanus  bacillus 532 

162.  Tetanus  bacillus, 532 

168.     Culture  of  Bacillus  tetani  in  nutrient  gelatin, 533 

164.  Bacillus  ocdematis  maligni, 538 

165.  Bacillus  cedematis  maligni, 538 

166.  Cultures  of.  Bacillus  cedematis  maligni  in  nutrient  gelatin,      .        .        .  539 
Hi?.     Bacillus  cadaveris, 541 

168.  Bacillus  cadaveris 541 

169.  Bacillus  of  symptomatic  anthrax, 542 

170.  Bacillus  of  symptomatic  anthrax,    ........  542 

171.  Culture  of  bacillus  of  symptomatic  anthrax, 543 

172.  Spirillum  Obermeieri, 550 

178.    Spirillum  Obermeieri, 550 

174.  Spirillum  choleras  Asiatic®, 552 

175.  Spirillum  cholerae  Asiaticae, :>.YJ 

176.  Colonies  of  Spirillum  cholera  Asiaticae 553 

177.  Spirillum  cholerae  Asiaticae, 553 

178.  Cultures  of  Spirillum  cholerae  Asiatic;r  in  nutrient  gelatin,     .        .        .  554 

179.  Spirillum  cholera;  Asiaticae, 554 

180.  Colonies  in  nutrient  gelatin  of  Spirillum  cholerae  Asiaticae,  Spirillum 

tyrogenum,  and  Spirillum  of  Finkler  and  Prior,         ....  555 

181.  Section  through  mucous  membrane  of  intestine  from  cholera  cadaver,  559 

188.    Spirillum  of  Finkl.-r  -md  Prior 562 

188.    Colonies  of  Spirillum  of  Finkler  and  Prior, 5r,-j 

184.    Spirillum  of  Finkler  and  Prior;  culture  in  gelatin,        ....  562 


LIST   OF  ILLUSTRATIONS.  xi 

FIG-  PAGE 

185.  Spirillum  tyrogenum, ,  553 

186.  Colonies  of  Spirillum  tyrogenum, 563 

187.  Spirillum  Metschnikovi, 564 

188.  Penicillum  glaucum,        ..........  624 

189.  Miquel's  aeroscope, 625 

190.  Hesse's  aeroscope, 627 

191.  Miquel's  flask, 629 

192.  Straus  and  Wiirtz's  soluble  filter, 629 

193.  Petri's  sand  filter, 630 

194.  Sugar  filter, 631 

195.  Sedgwick  and  Tucker's  apparatus, 631 

196.  Sternberg's  vacuum  tube, 637 

197.  Lepsius'  apparatus  for  collecting  water  at  various  depths,      .        .        .  638 

198.  Koch's  plate  method, 639 

199.  Smear  preparation  from  liver  of  yellow-fever  cadaver,    ....  675 

200.  Bacillus  cadaveris  grandis, 675 


PART    FIRST. 


CLASSIFICATION,   MORPHOLOGY,  AND  GENERAL 
BACTERIOLOGICAL  TECHNOLOGY. 

I.  HISTORICAL.    II.  CLASSIFICATION.    III.   MORPHOLOGY.     IV.  STAINING 
METHODS.    V.  CULTURE  MEDIA.     VI.  STERILIZATION  OP  CULTURE 
MEDIA.    VII.  CULTURES  IN  LIQUID  MEDIA.    VIII.  CULTURES 
IN  SOLID  MEDIA.    IX.  CULTIVATION  OF  ANAEROBIC  BAC- 
TERIA.   X.  INCUBATING  OVENS  AND  THERMO  REGU- 
LATORS.    XI.  EXPERIMENTS  UPON  ANIMALS. 
XII.   PHOTOGRAPHING  BACTERIA. 


PAET    FTEST. 


I. 

HISTORICAL. 

IT  is  probable  that  Leeuwenhoeck,  "  the  father  of  microscopy/' 
observed  some  of  the  larger  species  of  bacteria  in  faeces,  putrid  in- 
fusions, etc.,  which  he  examined  with  his  magnifying  glasses  (1675), 
but  it  was  nearly  a  century  later  before  an  attempt  was  made  to  de- 
fine the  characters  of  these  minute  organisms  and  to  classify  them 
(O.  F.  Miffler,  1773). 

In  the  absence  of  any  reliable  methods  for  obtaining  pure  cultures, 
it  is  not  surprising  that  the  earlier  botanists,  in  their  efforts  to  classify 
microorganisms,  fell  into  serious  errors,  one  of  which  was  to  include 
under  the  name  of  infusoria  various  motile  bacteria.  These  are  now 
generally  recognized  as  vegetable  organisms,  while  the  Infusoria  are 
unicellular  animal  organisms. 

Ehrenberg  (1838),  under  the  general  name  of  Vibrioniens,  de- 
scribes four  genera  of  filamentous  bacteria,  as  follows  : 

1.  Bacterium — filaments  linear  and  inflexible  ;  three  species. 

2.  Vibrio — filaments  linear,  snake-like,  flexible  ;  nine  species. 

3.  Spirillum — filaments  spiral,  inflexible  ;  three  species. 

4.  Spirochcete — filaments  spiral,  flexible  ;  one  species. 

These  vibrioniens  were  described  by  Ehrenberg  as  "  filiform  ani- 
mals, distinctly  or  apparently  polygastric,  naked,  without  external 
organs,  with  the  body  uniform  and  united  in  chains  or  in  filiform 
series  as  a  result  of  incomplete  division/' 

Dujardin  (1841)  also  placed  the  vibrioniens  of  Ehrenberg  among 
the  infusoria,  describing  them  as  "filiform animals,  extremely  slen- 
der, without  appreciable  organization,  and  without  visible  locomotive 
organs." 

Charles  Robin  (1853)  suggested  the  relationship  of  Ehrenberg's 
vibrioniens  with  the  genus  Leptothrix,  which  belongs  to  the  algae ; 
and  Davaine  (1859)  insisted  that  the  vibrioniens  are  vegetable  organ- 


HISTORICAL. 

nearly  allied  to  the  algae.  His  classification  will  be  found 
in  the  "  Dictionnaire  Encyclop.  des  Sciences  Medicales,"  art.  "  Bac- 
teriej*  "  (1868).  This  view  is  also  sustained  by  the  German  botanist 
Cohn  and  is  now  generally  accepted. 

Spallan/ani,  in  177»>,  endeavored  to  show  by  experiment  that  the 
generally  received  theory  of  the  spontaneous  generation  of  micro- 
organisms in  organic  liquids  was  not  true.  This  he  did  by  boiling 
putrescible  liquids  in  carefully  sealed  flasks.  The  experiment  was 
n  <>t  always  successful,  but  in  a  certain  number  of  instances  the 
liquids  were  sterilized  and  remained  unchanged  for  an  indefinite 
Period.  The  objection  was  raised  to  these  experiments  that  the  oxy- 
gen of  the  air  was  excluded  by  hermetically  sealing  the  flasks,  and 
it  was  claimed,  in  accordance  with  the  views  of  Gay-Lussac,  that 
free  admission  of  this  gas  was  essential  for  the  development  of  fer- 
mentation. 

This  objection  was  met  by  Franz  Schulze  (1836),  who  admitted  air 
to  boiled  putrescible  liquids  by  drawing  it  through  strong  sulphuric 
acid,  in  which  suspended  microorganisms  were  destroyed.  He  thus 
demonstrated  that  boiled  solutions,  which,  when  exposed  to  the  air 
without  any  precautions,  quickly  fell  into  putrefaction,  remained  un- 
changed when  freely  supplied  with  air  which  had  been  passed  through 
an  agent  capable  of  quickly  destroying  all  living  organisms  con- 
tain. M!  in  it. 

Schwann  (1839)  demonstrated  the  same  fact  by  another  method. 
Air  was  freely  admitted  to  his  boiled  liquids  through  a  tube  which 

->  heated  to  a  point  which  insured  the  destruction  of  suspended 
microorganisms.  The  same  author  is  entitled  to  the  credit  of  hav- 
ing first  clearly  stated  the  essential  relation  of  the  yeast  plant  — 
lioromyces  cereuisice  —  to  the  process  of  fermentation  in  saccha- 
rine liquids,  which  results  in  the  formation  of  alcohol  and  carbonic 
acid. 

Helmholtz,  in  1843,  repeated  the  experiments  of  Schwann  with 
calcined  air,  and  arrived  at  similar  results  —  i.e.,  he  found  that  the 
free  admission  of  calcined  air  to  boiled  organic  infusions  did  not  pro- 
<luct3  fermentation  of  any  kind. 

It  was  objected  to  these  experiments  that  the  air,  having  been 
^'ihject.-d  t"  ;|  hitfh  temperature,  had  perhaps  undergone  some  chem- 
i--.ll  change  which  prevented  it  from  ina  HI;-  lira  ting  processes  of  fer- 


This  objection  was  met  by  Schroder  and  Von  Dusch  (1854)  by  a 

\  simpl,.  device  which  has  since  proved  to  ho  of  inestimable  value 

in  bacteriological   researches,     These  observers  showed  that  a  loose 

plug  of  cotton,  through  which  free  communication  with  the  external 

air  is  maintained,  excludes  all   suspended  microorganisms,  and  that 


HISTORICAL.  5 

air  passed  through  such  a  filter  does  not  cause  the  fermentation  of 
boiled  organic  liquids. 

The  experiments  of  Pasteur  and  of  Hoffman,  made  a  few  years 
later,  showed  that  even  without  a  cotton  filter,  when  the  neck  of  the 
flask  containing  the  boiled  liquid  is  long  drawn  out  and  turned  down- 
ward, the  contents  may  be  preserved  indefinitely  without  change. 
In  this  case  suspended  particles  do  not  reach  the  interior  of  the  flask, 
as  there  is  no  current  of  air  to  carry  them  upward  through  its  long- 
drawn-out  neck,  and  they  are  prevented  by  the  force  of  gravity  from 
ascending. 

Tyndall  showed  at  a  later  date  that  in  a  closed  chamber,  in  which 
the  air  is  not  disturbed  by  currents,  all  suspended  particles  settle  to 
the  floor  of  the  chamber,  leaving  the  air  optically  pure,  as  is  proved 
by  passing  a  beam  of  light  through  such  a  chamber. 

Notwithstanding  the  fact  that  the  experimenters  mentioned  had 
succeeded  in  keeping  boiled  organic  liquids  sterile  in  flasks  to  which 
the  oxygen  of  the  air  had  free  access,  the  question  of  the  possibility 
of  spontaneous  generation — heterogenesis — still  remained  unsettled, 
inasmuch  as  occasionally  a  development  of  bacterial  organisms  did 
occur  in  such  boiled  liquids. 

This  fact  was  explained  by  Pasteur  (1860),  who  showed  that  the 
generally  received  idea  that  the  temperature  of  boiling  water  must 
destroy  all  living  organisms  was  a  mistaken  one,  and  that,  especially 
in  alkaline  liquids,  a  higher  temperature  was  required  to  insure  ster- 
ilization. His  experiments  showed  that  a  temperature  of  110°  to 
112°  C.  (230°  to  233.6°  F.),  which  he  obtained  by  boiling  under  a 
pressure  of  one  and  a  half  atmospheres,  was  sufficient  in  every  case. 
These  experiments,  which  have  been  repeated  by  numerous  investi- 
gators since,  settled  the  spontaneous-generation  controversy  ;  and  it 
is  now  generally  admitted  that  no  development  of  microorganisms 
occurs  in  organic  liquids,  and  no  processes  of  putrefaction  or  fermen- 
tation occur  in  such  liquids,  when  they  are  completely  sterilized  and 
guarded  against  the  entrance  of  living  germs  from  without. 

Pasteur  at  a  later  date  (1865)  showed  that  the  atmospheric  or- 
ganisms which  resist  the  boiling  temperature  are  in  fact  reproduc- 
tive bodies,  or  spores,  which  he  described  under  the  name  of  "  corpus- 
cles ovoides  "  or  "  corpuscles  brillants."  Spores  had  been  previously 
seen  by  Perty  (1852)  and  Robin  (1853),  but  it  was  not  until  1876  that 
the  development  of  these  reproductive  bodies  was  studied  with  care 
by  Cohn  and  by  Koch.  The  last-named  observer  determined  the 
conditions  under  which  spores  are  formed  by  the  anthrax  bacillus. 
Five  years  later  (1881)  Koch  published  his  valuable  researches  relat- 
ing to  the  resisting  power  of  anthrax  spores  to  heat  and  to  chemical 
agents. 


6  HISTORICAL. 

The  development  of  our  knowledge  relating  to  the  bacteria, 
stimulated  by  the  controversy  relating  to  spontaneous  generation 
and  by  the  demonstration  that  various  processes  of  fermentation 
and  putrefaction  are  due  to  microorganisms  of  this  class,  has 
depended  largely  upon  improvements  in  methods  of  research. 
Among  the  most  important  points  in  the  development  of  bacterio- 
logical technique  we  may  mention,  first,  the  use  of  a  cotton  air 
filter  (Schroder  and  Von  Dusch,  1854)  ;  second,  the  sterilization  of 
culture  fluids  by  heat  (methods  perfected  by  Pasteur,  Koch,  and 
others)  ;  third,  the  use  of  the  aniline  dyes  as  staining  agents  (first 
recommended  by  Weigert  in  1877)  ;  fourth,  the  introduction  of 
solid  culture  media,  and  the  "plate  method "  for  obtaining  pure  cul- 
tures, by  Koch  in  1881. 

The  various  improvements  in  methods  of  research,  and  espe- 
cially the  introduction  of  solid  culture  media  and  Koch's  "plate 
method"  for  isolating  bacteria  from  mixed  cultures,  have  placed 
bacteriology  upon  a  scientific  basis,  and  have  shown  that  many  of 
the  observations  and  inferences  of  the  earlier  investigators  were 
erroneous  owing  to  the  imperfection  of  the  methods  employed. 

Since  it  has  been  demonstrated  that  certain  infectious  diseases  of 
man  and  the  lower  animals  are  due  to  organisms  of  this  class,  phy- 
sicians have  been  especially  interested  in  bacteriological  researches, 
and  the  progress  made  during  the  past  fifteen  years  has  been  largely 
due  to  their  investigations.  It  was  a  distinguished  French  physi- 
cian, Davaine,  who  first  demonstrated  the  etiological  relation  of  a 
microorganism  of  this  class  to  a  specific  infectious  disease.  The  an- 
thrax bacillus  had  been  seen  in  the  blood  of  animals  dying  from  this 
disease  by  Pollender  in  1849  and  by  Davaine  in  1850,  but  it  was  sev- 
eral years  later  (1863)  before  the  last-named  observer  claimed  to 
have  demonstrated  by  inoculation  experiments  the  causal  relation  of 
the  bacillus  to  the  disease  in  question. 

The  experiments  of  Davaine  were  not  generally  accepted  as  con- 
clusive, because  in  inoculating  an  animal  with  blood  containing  the 
bacillus,  from  an  infected  animal  which  had  succumbed  to  the 
disease,  the  living  microorganism  was  associated  with  material 
ti"in  the  body  of  the  diseased  animal.  This  objection  was  subse- 
quently removed  by  the  experiments  of  Pasteur,  Koch,  and  many 
•  >t  IHTS  with  pure  cultures  of  the  bacillus,  which  were  shown  to  have 
the  same  pathogenic  effects  as  had  been  obtained  in  inoculation  ex- 
periments with  the  blood  of  an  infected  animal. 

The  next  demonstration  of  the  causal  relation  of  a  parasitic  mi- 
'Toorpmirtin  t<>  an  infectious  malady  was  made  by  Pasteur,  who  de- 
v..t,.,l  several  years  to  the  study  of  an  infectious  disease  of  silkworms 
threatened  to  destroy  the  silk  industry  of  France— pebrine. 


HISTORICAL.  7 

In  1873  Obermeier,  a  German  physician,  announced  the  discov- 
ery, in  the  blood  of  patients  suffering  from  relapsing  fever,  of  a  mi- 
nute, spiral,  actively  motile  microorganism — the  Spirochcete  Ober- 
meieri — which  is  now  generally  recognized  as  the  specific  infectious 
agent  in  this  disease. 

The  very  important  work  of  Koch  upon  traumatic  infectious 
diseases  was  published  in  1878. 

In  1879  Hansen  reported  the  discovery  of  bacilli  in  the  cells  of 
leprous  tubercles,  and  subsequent  researches  have  shown  that  this 
bacillus  is  constantly  associated  with  leprosy  and  presumably  bears 
an  etiological  relation  to  the  disease. 

In  the  same  year  (1879)  Neisser  discovered  the  "  gonococcus  "  in 
gonorrhoeal  pus. 

The  bacillus  of  typhoid  fever  was  first  observed  by  Eberth,  and 
independently  by  Koch,  in  1880,  but  it  was  not  until  1884  that  Gaff- 
ky's  important  researches  relating  to  this  bacillus  were  published. 

In  1880  Pasteur  published  his  memoir  upon  fowl  cholera,  and  the 
same  year  appeared  several  important  communications  from  this 
pioneer  in  bacteriological  research  upon  the  "attenuation"  of  the 
virus  of  anthrax  and  of  fowl  cholera  and  upon  protective  inocula- 
tions in  these  diseases. 

In  1880  the  present  writer  discovered  a  pathogenic  micrococcus, 
which  he  subsequently  named  Micrococcus  Pasteuri,  and  which  is 
now  generally  recognized  as  the  usual  agent  in  the  production  of 
acute  croupous  pneumonia — commonly  spoken  of  as  the  "  diplococ- 
cus  pneumoniae,"  but  described  in  the  present  volume  under  the 
name  of  Micrococcus  pneumonice  crouposce. 

In  1881  several  important  papers  by  Koch  and  his  colleagues  ap- 
peared in  the  first  volume  of  the  "  Mittheilungen  "  published  by  the 
Imperial  Board  of  Health  of  Germany. 

The  following  year  (1882)  Koch  published  his  discovery  of  the 
tubercle  bacillus. 

The  same  year  Pasteur  published  his  researches  upon  the  disease 
of  swine,  known  in  France  as  rouget. 

The  same  investigator  (Pasteur)  also  published  in  1882  his  first 
communication  upon  the  subject  of  rabies. 

Another  important  discovery  was  made  in  1882  by  the  German 
physicians  Loffler  and  Schiitz,  viz.,  that  of  the  bacillus  of  glan- 
ders. 

Koch  published  his  discovery  of  the  cholera  spirillum — "  comma 
bacillus  "—in  1884. 

The  same  year  (1884)  Loffler  discovered  the  diphtheria  bacillus. 

Another  important  publication  during  the  same  year  was  that  of 
Rosenbach,  who,  by  the  application  of  Koch's  methods,  fixed  defi- 


HISTORICAL. 

nit<-ly  the  characters  of  the  various  microorganisms  found  in  pus 
1 1 « .ni  acute  abscesses,  etc. 

The  tetanus  bacillus  was  discovered  in  1884  by  Nicolaier,  a  stu- 
dent in  the  laboratory  of  Prof.  Fliigge,  of  Gottingen.  That  this 
bacillus  is  the  cause  of  tetanus  in  man  has  been  demonstrated  by  the 
subsequent  researches  of  numerous  investigators.  For  an  exact  knowl- 
edge of  its  biological  characters  we  are  especially  indebted  to  Kitasato. 

So  far  as  human  pathology  is  concerned,  no  important  pathogenic 
microorganism  was  discovered  after  the  year  1884  until  the  year  1892. 
After  numerous  unsuccessful  researches  by  competent  bacteriologists, 
a  bacillus  was  discovered  by  Pfeiffer,  of  Berlin,  and  independently 
by  Canon,  which  is  believed  to  be  the  specific  cause  of  influenza. 

In  1894  the  distinguished  Japanese  bacteriologist,  Kitasato,  dur- 
ing a  visit  to  China  made  for  the  purpose,  discovered  the  bacillus 
of  the  bubonic  plague  of  the  Orient. 

Recent  experimental  evidence  appears  to  justify  the  conclusion 
that  infectious  pleuro-pneumonia  of  cattle  is  due  to  a  bacillus  dis- 
covered by  Arloing— his  Pneumobacillus  liquefaciens  bovis. 

Finally  we  may  refer  to  the  recent  discovery  of  the  antitoxins  of 
diphtheria  and  of  tetanus  as  one  of  the  most  important  events  in  the 
history  of  bacteriology  and  of  scientific  medicine.  The  name  of  Behr- 
ing  has  the  first  place  in  connection  with  this  discovery. 

Having  briefly  passed  in  review  some  of  the  principal  events  in 
the  progress  of  our  knowledge  in  this  department  of  scientific  investi- 
gation, it  will  be  of  interest  to  students  to  know  something  more  of 
the  literature  of  bacteriology.  Important  papers  have  appeared  in 
medical  and  scientific  journals  in  all  countries,  and  research  work  of 
value  has  been  done  by  enthusiastic  investigators  of  nearly  every 
nation.  The  brilliant  pioneer  work  done  by  Pasteur  and  by  Koch  has 
attracted  to  them  many  pupils  and  has  made  France  and  Germany 
the  leading  countries  in  this  line  of  investigation.  The  very  great 
advantages  of  Koch's  methods  of  research,  introduced  at  the  com- 
mencement of  the  last  decade,  have  attracted  many  students  from 
various  parts  of  the  world  to  Berlin,  and  to  other  cities  of  Germany 
where  instruction  was  to  be  obtained  from  some  of  Koch's  earlier 
pupils.  But  to-day  bacteriological  laboratories  have  been  established 
in  all  parts  of  the  world,  and  it  is  no  longer  necessary  to  go  to  Ger- 
many to  obtain  such  instruction.  The  literature  of  the  subject  is, 
however,  largely  in  the  German  and  French  languages.  We  can 
only  refer  here  to  such  periodicals  as  are  principally  devoted  to  bac- 
t.-riological  research  work. 

The  Zeitxchrift  ///>  Ifi/giene  has  been  published  since  1886,  and 
contains  numerous  valuable  papers,  contributed  for  the  most  part  by 
the  pupils  of  Koch  and  of  Fliigge,  who  are  the  editors  of  the  journal. 


HISTORICAL.  9 

The  Annales  de  VInstitut  Pasteur  is  a  monthly  journal  which 
has  been  published  since  1888.  It  is  edited  by  Duclaux,  and  contains 
many  important  papers  and  reviews,  as  well  as  the  statistics  of  the 
Pasteur  Institute  relating  to  preventive  inoculations  against  hydro- 
phobia. 

The  Annales  de  Micrography  is  a  monthly  journal,  published  in 
Paris.  The  principal  editor  is  Miquel. 

The  Centralblatt  fur  Bakteriologie  und  Parasitenkunde  is  a 
weekly  journal  which  has  been  published  by  Gustav  Fischer,  of  Jena, 
since  1887.  The  editors  are  Uhlworm,  of  Cassel;  Loffler,  at  present 
professor  at  Greifswald;  and  Leuckart,  professor  at  Leipzig. 

The  Journal  of  Pathology  and  Bacteriology  is  published 
monthly  in  Edinburgh  and  London.  It  dates  from  1892. 

A  most  important  work  for  students  of  bacteriology  is  the  Jahres- 
bericht  of  Baumgarten,  which  has  been  published  since  1885  by  Harald 
Bruhn,  Braunschweig,  Germany.  This  gives  a  brief  abstract  of 
nearly  every  paper  of  importance  relating  to  the  subject  which  has 
been  published  during  the  year. 


II. 

CLASSIFICATION. 

THE  earlier  naturalists— Ehrenberg  (1838),  Dujardin  (1841)— 
placed  the  bacteria  among  the  infusoria;  but  they  are  now  recog- 
nized as  vegetable  microorganisms,  differing  essentially  from  the 
infusoria,  which  are  unicellular  animal  organisms.  One  of  the  prin- 
cipal points  in  differentiating  animal  from  vegetable  organisms 
among  the  lowest  orders  of  living  things  is  the  fact  that  animal 
organisms  receive  food  particles  into  the  interior  of  the  body,  assimi- 
lating the  nutritious  portion  and  subsequently  extruding  the  non- 
nutritious  residue  ;  vegetable  organisms,  on  the  other  hand,  are 
nourished  through  the  cell  wall  which  encloses  their  protoplasm,  by 
organic  or  inorganic  substances  held  in  solution. 

Ehrenberg  (1838),  under  the  name  of  vibrioniens,  established  four  gen- 
era, as  follows: 

1.  Bacterium — filaments  linear  and  inflexible. 

2.  Vibrio — filaments  linear,  snake-like,  flexible. 

3.  Spirillum — filaments  spiral,  inflexible. 

4.  Spirochcete — filaments  spiral,  flexible. 

Duiardin  (1841)  united  the  two  genera  Spirillum  and  Spirochcete  of 
Ehrenberg,  and  added  to  the  description  of  the  generic  characters  as  fol- 
lows: 

1.  Bacterium — filaments  rigid,  with  a  vacillating  movement. 

2.  Vibrio — filaments  flexible,  with  an  undulatory  movement. 

3.  Spirillum — filaments  spiral,  movement  rotatorv. 

It  will  be  seen  that  this  classification  leaves  no  place  for  the  motionless 
bacilli,  such  as  the  anthrax  bacillus  and  many  others,  and  does  not  include 
the  spherical  bacteria,  now  called  micrococci. " 

The  classification  of  Davaine  (18t>8)  provides  for  the  motionless,  fila- 
m<  ntous  bacteria,  but  does  not  include  the  micrococci.  This  author  first 
insisted  that  the  vibrioniens  of  Ehrenberg  are  truly  vegetable  organisms, 
allied  to  the  algae.  He  makes  four  genera,  as  follows: 

Filaments    straight    or    bent,  (  Moving    spontane-  I  Rigid  Bacterium. 
but  not  in  a  spiral,  ously,  \  Flexible  Vibrio. 

( Motionless,        .          Bacteridium. 
Filaments  spiral, Spirillum. 

Following  Davaine,  the  French  bacteriologists  frequently  speak  of  the 
motionless  anthrax  bacillus  as  la  bacttridie. 

llotVinaii  in  1869  included  in  his  classification  the  spherical  bacteria, 
and  pointed  out  the  fact  that  motility  could  not  be  taken  as  a  generic  char- 
acter, us  it  was  not  constant  in  the  same  species  and  depended  to  some  ex- 
tent upon  temperature  conditions,  etc. 


CLASSIFICATION.  H 

Having  determined  that  the  bacteria  are  truly  vegetable  organ- 
isms, the  attention  of  botanists  has  been  given  to  the  question  as  to 
what  class  of  vegetable  organisms  they  are  most  nearly  related  to. 
There  are  decided  differences  of  opinion  in  this  regard.  While  Da- 
vaine,  Rabenhorst,  and  Cohn  insist  upon  their  affinities  with  the 
algae,  Robin,  Nageli,  and  others  consider  them  fungi.  One  of  the 
principal  characters  which  distinguish  the  algae  from  the  fungi  is 
the  presence  of  chlorophyll  in  the  former  and  its  absence  in  the  latter. 
Now,  the  bacteria  are  destitute  of  chlorophyll,  and  in  this  regard 
resemble  the  fungi;  yet  in  others  their  affinities  with  the  Palmellacece 
and  Oscillatoriacece  are  unmistakable.  It  is  not  necessary,  how- 
ever, that  we  should  consider  them  as  belonging  to  either  of  these 
classes  of  the  vegetable  kingdom.  By  considering  theni  a  distinct 
class  of  unicellular  vegetable  organisms,  under  the  general  name  of 
bacteria,  we  may  avoid  the  difficulties  into  which  the  botanists  have 
fallen. 

We  must  refer  briefly,  however,  to  the  classification  proposed  by  some 
of  the  leading  German  botanists. 

Nageli,  placing  the  bacteria  among  the  lower  fungi,  which  give  rise  to 
the  decomposition  of  organic  substances,  divides  these  into  three  groups : 

1.  The  Mucorini,  or  mould  fungi. 

2.  The  Saccharomycetes,  or  budding  fungi,  which  produce  alcoholic  fer- 
mentation in  saccharine  liquids. 

3.  The  Schizomycetes,  or  fission  fungi,  which  produce  putrefactive  pro- 
cesses, etc. 

Cohn,  under  the  name  of  Schizophytes,  has  grouped  these  low  vegetable 
organisms,  whether  provided  or  not  with  chlorophyll,  into  two  tribes  hav- 
ing the  following  characters : 

1.  GL.EOGENES— cells  free  or  united  into  glairy  families  by  an  intercel- 
lular substance. 

2.  NEMATOGENES— cells  disposed  in  filaments. 

In  the  first  tribe  he  has  placed  the  genera  Micrococcus  (Hallier),  Bacte- 
rium (Dujardin),  Merismopedia  (Meyer),  Sarcina  (Goodsir),  and  Ascococcus 
(Billroth),  with  various  genera  of  unicellular  algae  containing  chlorophyll. 

In  the  second  tribe  we  have  the  genera  Bacillus  (Cohn),  Leptothrix 
(Kg.),  Vibrio  (Ehr.),  Spirillum  (Ehr.),  Spirochcete  (Ehr.),  Streptococcus 
(Billr.),  Cladothrix  (Cohn),  and  Streptothrix  (Cohn),  associated  with  gen- 
era of  green  filamentous  algae. 

The  German  botanist  Sachs  unites  the  fungi  and  the  algae  into  a  single 
group,  the  Thallophytes,  in  which  he  establishes  two  parallel  series,  one  in- 
cluding those  containing  chlorophyll,  and  the  other  without,  as  follows: 


THALLOPHYTES. 

Forms  with  chlorophyll.  Forms  without  chlorophyll. 

Class!  I. — Protophytes. 

A.  Cyanophycese     (Oscillatoria-  A.  Schizomycetes  (Bacteria), 
ceae,  etc.). 

B.  Palmellacese.  B.  Saccharomycetes. 


12  CLASSIFICATION. 

Zopf.  who  insists  upon  the  polymorphism  of  these  low  organisms,  divides 
tho  h;trt«-n.-»  into  four  groups: 

Genera. 

Streptococcus, 

1.   O»rcocE,«.— Up   to  the    pre-  |          Merismopedia, 
sent  time,  only  known  in  the  form  of  \         Sarcina, 
cocci.  Micrococcus, 

Ascococcus. 


2.  BACTERIACE^E.— Have  for  the 
part    spherical,   rod-like,   and 
filamentous  forms  ;  the  first  (cocci) 


Bacterium, 

Spirillum, 

Vibrio, 


may  be  wanting  ;  the  last  are  not  j          Leuconostoc, 
different  at  the  two  extremities;  fila-  Bacillus, 

ments  straight  or  spiral.  Clostridium. 

:\.    LEPTOTRICHE.E.  —  Spherical,  ]         Crenothrix 
rod-shaped,  and  filamentous  forms;  Beaaiatoa  ' 

the  last  show  a  difference  between  the  Phragmidiothrix, 

two  extremities  ;   filaments  straight  |          Levtothrix 
<  u-s i >i ral ;  spore  formation  not  known.  J 

4.    CLADOTRICHE^E.  —  Spherical,  } 
rod-shaped,  filamentous,  and   spiral  | 
forms  ;  the  filamentous    form    pre-  |-          Cladothrix. 
sents  pseudo-branches  ;  spore  forma-  | 
tion  not  known. 

The  main  objection  to  this  classification  is  that  it  assumes  a  pleomorph- 
ism  for  the  bacteria  of  the  second  group — Bact^riaceae — which  has  only  been 
established  for  a  few  species,  and  which  appears  not  to  be  general  among  the 
rod  shaped  and  spiral  bacteria. 

De  I&arv  diviaes  the  bacteria  into  two  principal  groups,  one  including 
those  which  form  endospores,  and  the  other  those  which  are  reproduced  by 
arthrospores.  But  our  Knowledge  is  yet  too  imperfect  to  make  this  classifi- 
cation of  value,  and  the  same  may  be  said  of  Hueppe's  recent  attempt  at 
classification,  in  which  the  mode  of  reproduction  is  a  principal  feature. 

The  classification  of  Baumgarten  (1890)  appears  to  us  to  have 
more  practical  value,  and,  with  slight  modifications,  we  shall  adopt 
it  in  the  present  volume.  This  author  divides  the  bacteria  into  two 
principal  groups,  as  follows  : 

GROUP  I.  Species  relatively  monomorphous. 

GROUP  II.  Species  pleomorphous. 

I  lie   first  group  includes  the  micrococci,  the   bacilli,  and  the 

'  ilia;  the  second  group  the  spirulina  of  Hueppe,  leptotrichece 
'/"pf),  and  cl(ul<>trirln><t'. 

The  pleomorphous  species  described  by  Hauser  under  the  generic 
n;iine  Proteus  are  included  in  the  second  group  among  the  spirulina. 
In  the  present  volume  we  have  described  these  pleomorphous  species 
among  tin-  harilli. 

The  COCCI,  fa  tin-  classification  of  Haumgarten,  constitute  a  sin  gle 
genus  with  the  following  subgenera  :  1.  I ) i plococcus ;  2,  Strepto- 
M<Tisnn,/H'<li,i  (Xopf)— ••  Mcrista"  (Hueppe);  4,  Sar- 
<<""  ((i tor)  :  5,  Mn  -rut-nt-cus  ("  staphylococci  "). 

The    P,\(  n.i.i   ;,iv  included   in    a   single  genus  embracing  all   of 


CLASSIFICATION.  13 

those  species  which  only  form  rod-shaped  cells,  and  filaments  com- 
posed of  rod-like  segments ;  or  straight  filaments  not  distinctly  seg- 
mented, which  may  be  rigid  or  flexible. 

The  SPIRILLA  are  also  included  in  a  single  genus,  embracing  all 
of  those  species  in  which  the  filaments  are  spiral  in  form  and  the 
segments  more  or  less  spiral  or  "comma-shaped" — filaments  either 
rigid  or  flexible. 

This  simple  morphological  classification  of  the  monomorphous 
group  of  Baumgarten  corresponds  with  the  nomenclature  now  gene- 
rally in  use  among  bacteriologists.  We  speak  of  the  spherical  bac- 
teria as  cocci  or  as  micrococci,  of  the  rod-shaped  bacteria  as  bacilli, 
and  of  the  spiral  bacteria  as  spirilla. 

It  is  true,  however,  that  we  are  sometimes  embarrassed  to  decide 
whether  a  particular  microorganism  belongs  to  one  or  the  other  of 
these  morphological  groups  or  so-called  genera.^  Aihong  the  bacilli, 
for  exainple,  we  may  have,  in  the  same  pure  culture,  rods  of  very 
different  lengths,  some  being  so  short  that  if  alone  they  might  be 
taken  for  cocci,  while  others  may  have  grown  out  into  long  fila- 
ments. But  if  we  are  assured  that  the  culture  is  pure  the  presence 
of  rod  forms  establishes  the  diagnosis,  and  usually  the  cocci-like 
elements,  when  carefully  observed,  will  be  seen  to  be  somewhat 
longer  in  one  diameter  than  in  the  other.  The  German  bacterio- 
logists generally  insist  upon  placing  among  the  bacilli  all  straight  bac- 
teria in  which,  as  a  rule,  one  diameter  is  perceptibly  greater  than 
that  transverse  to  it ;  and  several  species  of  well-known  bacteria 
which  were  formerly  classed  as  micrococci  are  now  called  bacilli — 
e.g.,  Friedlander's  bacillus  ("pneumococcus"),  Bacillus prodigiosus. 

The  distinction  made  by  Cohn  and  others  between  the  genus 
Bacterium  (Duj.)  and  the  genus  Bacillus  (Cohn)  cannot  be  main- 
tained, inasmuch  as  we  may  have  short  rods  and  quite  long  fila- 
ments in  the  same  pure  culture  of  a  single  species ;  and,  again,  the 
character  upon  which  the  genus  Vibrio  (Ehr.)  was  established— 
viz.,  the  fact  that  the  filaments  are  flexible  and  the  movements 
sinuous — is  not  a  sufficient  generic  or  even  specific  character,  for  in 
a  pure  culture  there  may  be  short  rods  which  are  rigid,  and  long 
filaments  which  are  flexible  and  have  a  sinuous  movement.  We 
therefore  to-day  speak  of  all  the  elongated  forms  as  bacilli,  unless 
they  are  spiral  and  have  a  corkscrew-like  motion,  in  which  case  they 
are  known  as  spirilla. 

The  bacteria  are  also  classified  according  to  their  biological  char- 
acters, and  it  will  be  necessary  to  consider  the  various  modes  of 
grouping  them  from  different  points  of  view  other  than  that  which 
relates  to  their  form.  This  is  the  more  important  inasmuch  as  we 
are  not  able  to  differentiate  species  by  morphological  characters 


14  CLASSIFICATION. 

alone.  Thus,  for  example,  there  are  among  the  spherical  bacteria,  or 
miriH>cocci,  numerous  well-established  species  which  the  most  expert 
microscopist  could  not  differentiate  by  the  use  of  the  microscope 
alone  ;  the  same  is  true  of  the  rod-shaped  bacteria.  The  assump- 
tion often  made  by  investigators  who  are  not  sufficiently  impressed 
with  this  fact,  that  two  microorganisms  from  different  sources,  or 
even  from  the  same  source,  are  the  same  because  stained  prepara- 
tions examined  under  the  microscope  look  alike,  has  led  to  serious 
errors  and  to  much  confusion.  As  an  example  of  what  is  meant  we 
may  refer  to  the  pus  organisms.  Before  the  introduction  of  Koch's 
44 plate  method"  micrococci  had  been  observed  in  the  pus  of  acute 
abscesses.  Some  of  these  were  grouped  in  chains — streptococci— 
and  some  were  single,  or  in  pairs,  or  in  groups  of  four  ;  but  whether 
these  were  simply  different  modes  of  grouping  in  a  single  species,  or 
whether  the  chain  micrococci  represented  a  distinct  species,  was  not 
determined  with  certainty.  That  there  were  in  fact  four  or  more 
distinct  species  to  be  found  in  the  pus  of  acute  abscesses  was  not 
suspected  until  Rosenbach  and  Passet  demonstrated  that  this  is  the 
case,  and  showed  that  not  only  is  the  streptococcus  a  distinct  species, 
but  that  among  the  cocci  not  associated  in  chains  there  are  three 
species  which  are  to  be  distinguished  from  each  other  by  their  color 
when  grown  on  the  surface  of  a  solid  culture  medium.  One  of  these 
has  a  milk-white  color,  one  is  of  a  lemon-yellow  color,  while  the  third 

i  golden-yellow. 

Those  microorganisms  which  form  pigment  are  called  chromo- 
genes,  or  chromogenic ;  those  which  produce  fermentations  are 
spoken  of  as  zymogenes,  or  zymogenic  ;  those  which  give  rise  to  dis- 
ease processes  in  man  or  the  lower  animals  are  denominated  patho- 
genes,  or  pathogenic.  We  cannot,  however,  classify  bacteria  under 
the  three  headings  chromogenes,  zymogenes,  and  pathogenes,  for 
some  of  the  chromogenic  species  are  also  pathogenic,  as  are  some 
<>t  the  zymogenes.  These  characters  must  therefore  be  considered 
separately  as  regards  each  species,  and  in  studying  its  life  history  and 
•  I  languishing  characters  we  determine  whether  it  is  chromogenic 
or  non-chrontogenic ;  whether  it  produces  special  fermentations ; 
and  whether  it  is  or  is  not  pathogenic  when  inoculated  into  the 
1  "Wl ir  Animals.  In  making  the  distinction  between  pathogenic 
•  M.I  non-pathogenic  microorganisms  we  must  remember  that  a 
certain  species  may  be  pathogenic  for  one  animal  and  not  for  an- 
other. Thus  the  anthrax  bacillus,  which  is  fatal  to  cattle,  sheep, 
rabbits,  guinea-pigs,  and  mice,  does  not  kill  white  rats ;  the  bacillus 
of  mouse  septicaemia  kills  house  mice,  but  field  mice  are  fully  im- 
mune from  its  pathogenic  effects ;  on  the  other  hand,  the  bacillus  of 
glanders  is  fatal  to  field  mice  but  not  to  house  mice. 


CLASSIFICATION.  15 

Again,  it  must  be  remembered  that  pathogenic  power  also  de- 
pends, to  a  greater  or  less  extent,  upon  the  dose  injected  into  an 
animal  as  compared  to  its  body  weight.  Some  pathogenic  organ- 
isms in  very  minute  doses  give  rise  to  a  fatal  infectious  malady ; 
others  are  only  able  to  overcome  the  vital  resisting  power  of  the 
tissues  and  fluids  of  the  body  when  introduced  into  the  circulation, 
or  into  the  subcutaneous  tissue  or  abdominal  cavity,  in  considerable 
amounts.  Some  pathogenic  bacteria  invade  the  blood  ;  others  mul- 
tiply only  in  certain  tissues  of  the  body  ;  and  others  again  multiply 
in  the  intestine  and  by  the  formation  of  poisonous  products  which 
are  absorbed  show  their  pathogenic  power. 

Another  classification  of  the  bacteria  relates  to  the  environment 
favorable  to  their  development.  Thus  we  speak  of  saprophytic  and 
parasitic  bacteria,  or  of  SAPROPHYTES  and  PARASITES. 

The  saprophytes  are  such  as  exist  independently  of  a  living  host, 
obtaining  their  supply  of  nutriment  from  dead  animal  or  vegetable 
material  and  from  water  containing  organic  and  inorganic  matters 
in  solution.  The  strict  parasites,  on  the  other  hand,  depend  upon 
a  living  host,  in  the  body  of  which  they  multiply,  sometimes  without 
injury  to  the  animal  upon  which  they  depend  for  their  existence,  but 
frequently  as  harmful  invaders  giving  rise  to  acute  or  chronic  infec- 
tious diseases.  Microorganisms  which  ordinarily  lead  a  saprophy- 
tic existence,  but  which  can  also  thrive  within  the  body  of  a  living 
animal,  are  called  facultative  parasites.  Thus  the  leprosy  bacillus, 
which  is  only  found  in  leprous  tissues,  is  a  strict  parasite  ;  while  the 
typhoid  bacillus,  the  cholera  spirillum,  etc. ,  are  facultative  parasites, 
inasmuch  as  they  are  capable  of  maintaining  an  independent  exist- 
ence, for  a  time  at  least,  external  to  the  bodies  of  living  animals. 

It  seems  probable  that  the  pathogenic  organisms  which  are  only 
known  to  us  to-day  as  strict  parasites  were,  at  some  time  in  the  past, 
saprophytes,  which  gradually  became  accustomed  to  a  parasitic 
mode  of  existence,  and,  under  the  changed  conditions  of  their  envi- 
ronment, finally  lost  the  power  of  living  in  association  with  other 
saprophytes  exposed  to  variations  of  temperature,  etc.  The  tubercle 
bacillus,  for  example,  is  known  to  us  only  as  a  parasite  which  has  its 
habitat  in  the  lungs,  lymphatic  glands,  etc. ,  of  man  and  of  certain 
of  the  lower  animals.  But  we  are  able  to  cultivate  it  in  artificial 
media  external  to  the  body  ;  and  it  is  in  accord  with  modern  views 
relating  to  the  development  of  species  to  suppose  that  at  some  time 
in  the  past  it  was  able  to  lead  a  saprophytic  existence.  Not  to  admit 
this  forces  us  to  the  conclusion  that,  at  some  time  subsequent  to  the 
appearance  of  man  and  the  lower  animals  in  which  it  is  now  found 
as  a  parasite,  it  was  created  with  its  present  biological  characters, 
which  restrict  it  to  a  parasitic  existence  in  the  bodies  of  these  ani- 


],;  CLASSIFICATION. 

mals.  and  that,  consequent!}',  the  immense  destruction  of  human  life 
\vhidi  has  resulted  from  its  parasitic  invasion  of  successive  genera- 
tions was  designed  when  it  was  created.  The  opposite  view  is  sup- 
ported by  numerous  facts  which  show  that  these  low  organisms,  like 
those  higher  in  the  scale,  are  subject  to  modifications  as  a  result  of 
changed  conditions  of  environment,  and  that  such  modifications,  in 
the  course  of  time,  may  become  well-established  specific  characters. 

Again,  the  bacteria  may  be  grouped  into  aerobic  and  anaerobic 
species.  This  is  a  very  important  distinction,  which  was  first  estab- 
lish. M!  by  Pasteur,  who  found  that  certain  bacteria  will  only  grow 
when  freely  supplied  with  oxygen,  while  others  absolutely  decline  to 
grow  in  the  presence  of  this  gas.  The  latter,  which  are  spoken  of  as 
.s///V/  (tuaerobics,  maybe  cultivated  in  a  vacuum  or  in  an  atmo- 
sphere of  hydrogen.  Those  species  which  grow  either  in  the  pre- 
sence of  oxygen  or  when  it  is  excluded  are  called  facultative  an- 
a&robics, 

Certain  bacteria  produce  a  peptonizing  ferment  which  has  the 
power  of  liquefying  gelatin.  This  has  led  to  the  classification  of 
those  microorganisms  of  this  class  which  grow  in  Koch's  flesh-pep- 
tone-gelatin as  liquefying  and  non-liquefying  bacteria. 

Again,  we  speak  of  them  as  motile  or  non-motile. 

It  is  evident  that  these  biological  characters,  although  all-im- 
portant in  the  definition  of  species,  cannot  serve  us  in  an  attempt  to 
establish  natural  genera  ;  for  the  lines  are  not  sharply  drawn  between 
the  saprophytes  and  the  parasites,  the  aerobics  and  the  anaerobics, 
etc. ,  inasmuch  as  we  have  facultative  parasites  and  facultative  an- 
aerobics which  we  cannot  include  in  either  class,  and  which  yet  do 
not  form  a  distinct  class  by  themselves.  We  therefore  adhere  to  the 
morphological  classification,  although  this  is  open  to  criticism.  For 
example,  among  the  rod-shaped  organisms  which  we  call  bacilli  and 
describe  under  the  generic  name  Bacillus  there  are  some  which 
multiply  by  binary  division  only,  while  others  form  endogenous  re- 
productive bodies  known  as  spores.  Certainly  so  important  a  differ- 
ence in  the  mode  of  reproduction  should  be  sufficient  to  separate 
these  rod-shaped  organisms  into  two  natural  groups  or  genera. 

As  heretofore  stated,  the  German  bacteriologist  Hueppe  has  at- 
t'  mpted  a  classification  based  upon  the  mode  of  reproduction,  in 
which  h<-  makes  two  groups,  or  "tribes,"  one  in  which  reproduction 
occurs  by  tl m  formation  of  endogenous  spores— "  endospores  "— the 
other  in  which  it  occurs  by  the  formation  of  "  urtliroworrx."  '  The 
latter  group  includes  all  of  those  bacteria  in  which  no  other  mode  of 
multiplication  is  known  than  that  by  binary  division,  which  is  com- 
111011  to  ;l11  ln  tin-  present  state  of  our  knowledge  this  classification 
1  An  account  of  this  mode  of  reproduction  is  given  on  page  19. 


CLASSIFICATION.  17 

is  scarcely  to  be  considered  of  practical  value,  inasmuch  as  the  ques- 
tion of  spore  formation  is  still  undetermined  for  a  large  number  of 
species. 

In  the  following  table  we  shall  give  the  characters  of  the  dif- 
ferent genera  which  have  been  described  by  recent  botanists  and 
bacteriologists,  arranged  under  the  three  headings,  MICROCOCCI, 
BACILLI,  SPIRILLA.  Where  we  doubt  the  propriety  of  maintaining 
a  distinct  generic  name  upon  the  supposed  distinguishing  characters, 
the  description  will  be  printed  in  small  type. 

MICROCOCCI. 

General  Characters. — Spherical  bacteria  which  are  reproduced 
by  binary  division  ;  usually  without  spontaneous  movements  ;  do  not 
form  endogenous  spores.  (According  to  some  authors,  certain  cells, 
known  as  arthrospores,  may  be  distinguished  by  their  greater  size 
and  refractive  power,  and  these  are  supposed  to  have  greater  resist- 
ance to  desiccation  than  the  ordinary  cocci  resulting  from  binary 
division,  and  to  serve  as  reproductive  bodies.)  Some  micrococci  are 
not  precisely  round,  but  are  somewhat  oval  in  form ;  and  when  in 
process  of  division  the  cocci,  necessarily,  are  more  or  less  elongated 
in  one  diameter  before  a  complete  separation  into  two  spherical  ele- 
ments has  occurred. 

MiCROCOCCUS. — Division  in  one  direction  ;  cocci  single,  in  pairs, 
or  accidentally  associated  in  irregular  groups  ;  sometimes  held  to- 
gether in  irregular  masses  by  a  transparent,  glutinous,  intercellular 
substance.  (Micrococci  belonging  to  this  genus  are  frequently  de- 
scribed as  "  staphylococci,"  and  Staphylococcus  is  used  by  Rosen- 
bach  as  a  generic  name  for  the  pus  cocci  described  by  him,  which 
are  solitary  or  associated  in  irregular  groups,  as  above  described. ) 

Ascococcus. — Cocci  associated  in  globular  or  lobulated,  zoogloea 
masses  by  a  rather  firm  intercellular  substance. 

LEUCONOSTOC. — Cocci,  solitary  or  in  chains,  surrounded  by  a 
thick,  gelatinous  envelope  and  forming  zoogloea  of  cartilaginous 
consistence. 

STREPTOCOCCUS. — Division  in  one  direction  only ;  cocci  associ- 
ated in  chains. 

Diplococcus. — Division  in  one  direction  only  ;  cocci  associated  in  pairs. 

Association  in  pairs  is  common  to  all  of  the  micrococci,  inasmuch  as 
they  multiply  by  binary  division.  When  such  association  has  rather  a  per- 
manent character,  it  is  customary  to  speak  of  the  microorganism  as  a  diplo- 
coccus,  but  we  doubt  the  propriety  of  recognizing  this  mode  of  association 
as  a  generic  character. 

MERISMOPEDIA. — Division  in  two  directions,  forming  groups  of 
four,  which  remain  associated  in  a  single  plane — "tetrads." 

SARCINA. — Division  in  three  directions,  forming  packets  of  eight 


jg  CLASSIFICATION. 

or  more  elements,  which  remain  associated  in  more  or  less  regular 
cubical  masses. 


BACILLI. 


General  Characters.— Rod-shaped  and  filamentous  (not  spiral) 
bacteria  in  which  there  is  no  differentiation  between  the  extremities 
of  the  rods ;  reproduction  by  binary  division  in  a  direction  trans- 
verse to  the  long  axis  of  the  rods,  or  by  binary  division  and  the  for- 
mation of  endogenous  spores  ;  rigid  or  flexible  ;  motile  or  non-motile. 

BACILLUS. — Characters  as  given  above. 

Bacterium.—  This  genus,  established  by  Dujardin,  is  now  generally 
abandoned,  the  species  formerly  included  in  it  being  transferred  to  the  genus 
Bacillus.  As  defined  by  Cohn,  the  generic  characters  were  :  Cells  cylindri- 
cal or  elliptical,  free  or  united  in  pairs  during  their  division,  rarely  in 
fours,  never  in  chains,  sometimes  in  zooglcea  (differing  from  the  zooglcea 
of  spherical  bacteria  by  a  more  abundant  and  firmer  intercelluar  substance), 
having  spontaneous  movements,  oscillatory  and  very  active,  especially  in 
media  rich  in  alimentary  material  and  in  presence  of  oxygen. 

Clostridium.— Rod-shaped  bacteria  which  form  large,  endogenous,  and 
usually  oval  spores  ;  these  are  centrally  located,  and  during  the  stage  of 
spore  formation  the  rods  become  fusiform. 

SPIRILLA. 

General  Characters. — Curved  rods  or  spiral  filaments ;  rigid  or 
flexible  ;  reproduction  by  binary  division,  or  by  binary  division  and 
the  formation  of  endogenous  spores  (or  by  arthrospores  ?) ;  move- 
ments rotatory  in  the  direction  of  the  long  axis  of  the  filaments. 

SPIRILLUM. — Characters  as  above. 

Spirochcete.—  Flexible,  spiral  filaments;  movements  rotatory. 

Vibrio. — Filaments  flexible,  straight  or  sinuous;  movements  sinuous. 

A  considerable  number  of  bacteria  which  are  usually  seen  as  short,  curved 
rods,  but  which  may  grow  out  into  long,  spiral  filaments,  are  described  by 
so  MM-  authors  under  the  generic  name  Vibrio,  e.o.,  the  so-called  "comma 
bacillus"  of  Koch—"  Spirillum  cholerae  Asiatic*'  ;  the  spirillum  of  Finkler 
and  Prior — "  Vibrio  proteus" ;  the  spirillum  described  by  Gameleia — "  Vibrio 
M'  tschnikovi/'etc.  These  microorganisms  have  not  the  characters  which 
distin^ruislK'd  the  genus  Vibrio  as  established  by  Ehrenberg,  and  we  prefer  to 
follow  Fliigge  in  describing  them  under  the  generic  name  Spirillum. 

The  pathogenic  bacteria  now  known  belong  to  one  or  the  other 
of  the  above-described  genera,  and  the  attention  of  bacteriologists 
has  been  given  chiefly  to  the  study  of  micrococci,  bacilli,  and  spirilla. 
But  the  botanists  place  among  the  bacteria  certain  other  forms  which 
are  found  in  water,  and  which,  in  a  systematic  account  of  this  class 
of  microorganisms,  demand  brief  attention  at  least.  These  are  in- 
cluded in  Baumgarten's  second  group,  which  includes  the  pleomor- 
phous  bacteria. 

Spun  LIN  A  (Hueppe). — The  vegetative  cells  are  sometimes  rod- 
shaiH.«d  and  sometimes  spiral ;  in  suitable  media  they  may  grow  out 


CLASSIFICATION.  19 

into  long,  straight,  wavy,  or  spiral  filaments.  These  filaments  may 
break  up  into  cocci-like  reproductive  elements — "  arthrospores. " 

LEPTOTRICHE^E  (Zopf). — The  vegetative  cells  present  rod-shaped 
and  spiral  forms,  and  grow  out  into  straight,  wavy,  or  spiral  fila- 
ments ;  these  may  show  a  difference  between  the  two  extremities, 
of  base  and  apex.  Cocci-like  reproductive  bodies  are  formed  by  seg- 
mentation of  the  rod-shaped  elements  in  these  filaments.  In  some 
of  the  species  the  segments  are  enclosed  in  a  common  sheath.  Sub- 
genera:  LEPTOTHRIX,  BEGGIATOA,  CRENOTHRIX,  PHRAGMIDIO- 
THRIX  (for  generic  characters  see  page  12). 

CLADOTRICHE^E  (Zopf). — The  vegetative  cells  are  rod-shaped 
or  spiral,  and  grow  out  into  straight  or  spiral  filaments,  which  may 
present  pseudo-ramifications.  A  single  genus,  CLADOTHRIX  (see 
page  12). 


III. 

MORPHOLOGY. 

IN  the  present  chapter  we  shall  give  a  general  account  of  iiu? 
morphology,  modes  of  grouping,  and  dimensions  of  the  bacteria. 

The  standard  of  measurement  used  by  bacteriologists  is  the  micro- 
millimetre,  or  the  one-thousandth  part  of  a  millimetre.  This  is 
represented  by  the  Greek  letter  ^.  One  /*  (micromillimetre)  is  equal 
to  about  one-twenty-five-thousandth  of  an  English  inch. 

The  spherical  bacteria,  or  micrococci,  differ  greatly  in  size,  and 
also  in  the  mode  of  grouping  when,  as  a  result  of  binary  division, 
they  remain  associated  one  with  another.  The  smallest  may  mea- 
sure no  more  than  O.l/*,  while  some  of  the  larger  species  are  from 
one  to  two  p  in  diameter.  The  enormous  number  of  these  minute 
organisms  which  may  be  contained  in  a  small  drop  of  a  pure  culture 
may  be  easily  estimated  in  a  rough  way.  Compare  a  single  micro- 
coccus,  for  example,  with  a  sphere  having  a  diameter  of  one-twenty- 
fifth  of  an  inch.  If  our  micrococcus  is  one  of  the  larger  sort,  having 
a  diameter  of  one  //,  it  would  take  a  chain  of  one  thousand  to  reach 
across  the  diameter  of  such  a  sphere,  and  its  mass,  as  compared 
to  the  larger  sphere,  would  be  as  1  to  523,600,000. 

The  number  of  cocci  in  a  milligramme  of  a  pure  culture  of  Staphy- 
lococcus  pyogenes  aureus  has  been  estimated  by  Bujwid,  by  count- 
ing, at  8,000,000,000. 

Not  only  do  different  species  differ  in  dimensions,  but  consider- 
able differences  in  size  may  be  recognized  in  the  individual  cocci  in  a 
pure  culture  of  the  same  species.  On  the  other  hand,  there  are 
numerous  species  which  so  closely  resemble  each  other  in  size  and 
mode  of  association  that  they  cannot  be  differentiated  by  a  micro- 
scopic examination  alone,  and  we  must  depend  upon  other  characters, 
such  an  color,  growth  in  various  culture  media,  pathogenic  power, 
etc.,  to  decide  the  question  of  identity  or  non-identity. 

When  in  active  growth  the  micrococci  necessarily  depart  from  a 
typical  spherical  form  just  before  dividing,  and  under  these  circum- 
stanrrs  may  U'of  a  short  or  long  oval.  When  division  lias  taken 
place,  if  the  two  members  of  a  pair  remain  associated  they  are  often 
mure  or  less  flattened  at  the  jHniit  of  contact  (Fig.  1,  a). 


MORPHOLOGY. 


21 


When  in  a  culture  the  cocci  are  for  the  most  part  associated  in 
pairs  (Fig.  1,  d ) ,  we  speak  of  the  organism  as  a  diplococcus. 

The  staphylococci  are  characterized  by  the  fact  that,  for  the  most 
part,  the  individual  cocci  in  a  culture  are  solitary  (Fig.  1,  b).  But, 
inasmuch  as  multiplication  occurs  by  binary  division,  we  also  have 
pairs  and  occasionally  a  group  of  four — probably  from  the  accidental 
apposition  of  two  pairs  (Fig.  1,  c) ;  or  they  may  be  associated  in  grape- 

00 


oo 

80 


FIG.  1. 


like  bunches ;  and  after  staining  and  mounting  a  preparation  we  find 
the  cells  associated  in  irregular  groups.  This  results  from  the  fact 
that  they  are  surrounded  by  a  glutinous  material  which  causes  them 
to  adhere  to  each  other  (Fig.  1,  e).  A  mass  of  cocci  held  together  in 


Fio.  2. 


FIG.  3. 


FIG.  4. 


this  way  by  a  transparent,  glutinous,  intercellular  substance  is  spoken 
of  as  a  zooglcea  (Fig.  2).  In  the  genus  Ascococcus  the  intercellar 
substance  is  quite  firm  and  the  zooglcea  are  in  the  form  of  spherical 
or  irregularly  lobulated  masses  surrounded  by  a  resistant  envelope  of 
jelly-like  material  (Fig.  3). 

When,  as  a  result  of  division  in  one  direction  only,  the  cocci 


22  MORPHOLOGY. 

remain  united  in  chains  (Fig.  4,  a),  they  are  described  as  streptococci, 
and  are  sometimes  spoken  of  as  in  chaplets  or  in  torula  chains.  In 
such  chains  we  frequently  find  the  evidence  of  recent  division  of  the 
cocci,  as  shown  by  the  grouping  of  the  elements  of  the  chain  into 
pairs  (Fig.  4,  b). 

When  division  occurs  habitually  in  two  directions,  groups  of  four 
result,  which  are  spoken  of  as  tetrads.  This  is  the  distinguishing 
character  of  the  genus  Merismopedia.  In  these  groups  of  four  the 
individual  cocci  are  often  flattened  at  the  points  of  contact,  as  in 
Fig.  5,  6.  We  also  find  pairs  and  groups  of  three  in  pure  cultures  of 
species  belonging  to  this  genus,  as  shown  in  Fig.  5,  c.  In  these, 
transverse  division  has  not  yet  occurred  in  one  or  in  both  elements  of 
a  pair.  This  association  of  micrococci  in  tetrads  seems  to  be  main- 
tained, in  some  species  at  least,  by  the  fact  that  each  group  of  four  is 
enclosed  in  a  jelly-like  capsule.  The  extent  of  this  capsule  differs  in 
the  same  species  under  different  circumstances;  as  a  rule,  it  is  most 
apparent  when  a  culture  has  been  made  in  a  liquid  medium.  Some  of 

SB  88       OQ 

on  rjf) 

CD  **f 


00  SB 

e 
Fio.  5. 

the  diplococci  have  a  similar  capsule.  The  jelly-like  substance  does 
not  stain  well  with  the  aniline  colors  and  is  seen  as  a  transparent 
halo  around  the  stained  cocci.  Some  authors  (Frankel  and  Pfeiffer) 
believe  that  this  capsule  is  formed  by  the  swelling  up  of  the  cell 
membrane  as  a  result  of  the  imbibition  of  water. 

When  division  occurs  in  three  directions  packets  of  eight  or 
more  elements  are  formed.  This  mode  of  association  characterizes 
the  genus  Sarcina.  The  "packet  form  "is  best  seen  in  an  un- 
stained preparation  from  a  fresh  culture,  in  which  a  little  material 
>u>l»«'Mil.Ml  in  water  is  examined  under  a  comparatively  low-power 
objective — one-sixth  (Fig.  G). 

Among  the  bacilli  there  is  room  for  a  wider  range  of  morphologi- 
cal characters.  They  differ  not  only  in  dimensions  and  in  modes  of 
grouping,  but  in  form.  The  relation  of  the  transverse  to  the  longi- 
tudinal  diameters  affords  a  great  variety  of  forms,  varying  from  a 
short  oval  element  to  a  slender  rod  or  elongated  filament.  But  it 
must  he  remembered  that  \ve  may  h;ive  short  rods  and  long  filaments 
in  a  pure  culture  of  the  same  bacillus— the  typhoid  bacillus,  for 


MORPHOLOGY.  23 

example.  There  are  also  considerable  differences  in  the  transverse 
diameter  of  bacilli  belonging  to  the  same  species  when  cultivated  in 
different  media,  or  even  in  the  same  medium,  although,  as  a  rule, 
the  transverse  diameter  is  tolerably  uniform  in  pure  cultures. 

Again,  the  form  of  the  extremities  of  the  rods  is  to  be  observed 
(Fig.  7).  This  may  be  square,  or  the  corners  may  be  slightly 
rounded,  or  the  extremities  may  be  quite  round  or  lance-oval,  or 
the  outlines  of  the  rod  may  be  spindle-shaped  from  the  formation  of 


OO  c=x=>   00    CO 


a  large  central  spore — "clostridium" — or  one  end  may  be  dilated 
from  the  formation  of  a  large  terminal  spore. 

In  old  cultures  we  frequently  find  irregular  forms  due  to  swellings 
and  constrictions,  which  probably  occur  in  bacilli  which  have  but 
little  vitality  or  are  already  dead.  These  are  spoken  of  as  involution 
forms  (Fig.  8). 

The  bacilli  multiply  by  binary  division  in  a  direction  transverse 
to  the  longitudinal  axis,  and,  as  a  result  of  such  binary  division,  long 


Fio.  9. 

chains  in  which  the  elements  remain  associated  may  be  formed 
(Fig.  9)  ;  or  the  rods  may  be  for  the  most  part  solitary  or  united  in 
pairs.  Like  the  micrococci,  the  bacilli  are  sometimes  surrounded  by 
a  gelatinous  envelope  or  capsule.  They  may  also  be  united  by  a 
glutinous  material  into  zoogloea  masses. 

Bacilli  which  under  certain  conditions  are  seen  as  short  rods 
may,  under  other  circumstances,  grow  out  into  long  filaments,  and 
these  may  be  associated  in  bundles  or  in  tangled  masses. 

The  spirilla  differ  from  the  bacilli  in  the  form  of  the  rods  and  fila- 


24 


MORPHOLOGY. 


mente,  which  are  curved  or  spiral.  The  shorter  elements  in  a  pure 
culture  may  be  simply  curved,  as  in  a,  Fig.  10,  while  the  spiral  form 
becomes  apparent  in  those  which  are  longer,  and  we  may  have  one 
or  several  turns  of  the  spiral  (Fig.  10,  b).  The  spiral  form  may  be 
but  slightly  marked  (Fig.  10,  c),  or  the  turns  may  be  close  and  deep 
as  in  a  corkscrew  (Fig.  10,  d).  Again,  the  curved  filaments  may  be 
short  and  rigid,  or  long  and  flexible  (Fig.  10,  e). 

In  the  genus  Cladothrix,  which  is  placed  by  botanists  among 
the  bacteria,  the  filaments  appear  to  branch  ;  but  this  branching  is 
only  apparent,  and  there  is  no  true  dichotomous  branching  in  this 
class  of  microorganisms.  The  false  branching  of  Cladothrix 
<1n'hotoma,  Cohn,  is  shown  in  Fig.  11.  The  fact  that  some  of  the 
larger  species  of  bacilli  and  spirilla  are  provided  with  slender,  whip- 
like  appendages  called  flagella  has  been  known  for  many  years,  and 
it  has  for  some  time  been  suspected  that  all  of  the  motile  organisms 


Fio.  10. 


FIG. 11. 


Fio.  12. 


of  this  class  are  provided  with  similar  appendages  and  that  these  are 
organs  of  locomotion.  Recently,  by  improvements  in  methods  of 
staining,  Loftier  has  demonstrated  the  presence  of  flagella  in  many 
species  in  which  they  had  heretofore  escaped  observation.  They  are 
sometimes  single,  at  the  ends  of  the  rods  (Fig.  12,  a);  or  there  may 
be  several  at  the  extremity  of  a  single  rod  (Fig.  12,  &);  again,  they 
are  seen  in  considerable  numbers  around  the  periphery  of  the  rod 
(Fig.  12,  c). 

The  bacilli  and  spirilla  sometimes  contain  in  the  interior  of  the 
cells  granules  of  different  kinds.  These  may  appear  like  little  oil 
tJ  n  >ps  or  they  may  be  more  opaque.  In  the  genus  Beggiatoa  grains 
of  sulphur  are  found  in  the  interior  of  the  cells.  Again,  we  may 
find  vacuoles  in  the  protoplasm  ;  or,  in  stained  preparations,  deeply 
M;tin»-<I  <_rr;mulcs,  which  are  not  spores,  may  be  seen  at  the  extremi- 
ties of  the  rods — end-staining.  The  morphological  characters  de- 
IM ndiiiLC  upon  the  formation  of  endogenous  spores  will  be  referred  to 
hereafter. 


IV. 

STAINING  METHODS. 

THE  rapid  development  of  our  knowledge  with  reference  to  the 
minute  microorganisms  under  consideration  depends  very  largely 
upon  the  discovery  that  they  may  he  stained  by  various  dyes,  and  es- 
pecially by  the  aniline  colors.  Weigert  (1876)  was  the  first  to  employ 
these  colors  in  studying  the  bacteria,  and  Koch  at  once  recognized 
the  value  of  the  method  and  made  use  of  it  in  his  researches. 

The  basic  aniline  colors  are  those  employed,  and  among  these  the 
most  useful  are  fuchsin,  methylene  blue,  gentian  violet,  Bismarck 
brown,  and  vesuvin. 

Staining  upon  the  Cover  Glass  or  Slide. — By  a  "  cover-glass 
preparation "  we  mean  that  material  supposed  to  contain  bacteria 
has  been  spread  out  upon  a  thin  glass  cover,  dried,  and  stained  for 
microscopical  examination.  A  small  drop  of  a  liquid  culture  may,  for 


FIG.  13. 

example,  be  spread  upon  a  perfectly  clean  cover  glass  by  means  of  a 
platinum  wire  held  in  a  glass  handle  (Fig.  13).  Or  we  may  place  a 
drop  of  water  in  the  centre  of  the  thin  glass  cover,  and  by  means  of 
the  same  instrument  take  a  little  material  from  a  culture  made  upon 
the  surface  of  a  solid  medium  and  distribute  it  through  the  drop. 
In  this  case  we  must  be  careful  to  take  very  little  of  the  material,  as 
the  smallest  quantity  will  contain  an  immense  number  of  bacteria, 
and  for  a  satisfactory  view  of  the  individual  cells  it  is  necessary  that 
they  be  well  separated  from  each  other,  in  some  parts  of  the  prepa- 
ration at  least,  and  not  massed  together. 

Where  the  object  is  to  make  a  cabinet  preparation  for  permanent 
preservation,  special  care  should  be  taken  to  distribute  the  bacteria 
uniformly  through  the  drop  of  water.  The  next  step  consists  in  eva- 
porating the  liquid  so  that  the  bacteria  may  remain  attached  to  the 
surface  of  the  glass  cover.  This  may  be  done  by  simple  exposure  to 
the  air  or  by  the  application  of  gentle  heat.  When  the  bacteria  are 


26  STAINING  METHODS. 

suspended  in  an  albuminous  medium  it  will  be  necessary,  after  the 
Him  is  dry.  to  hfat  tlu>  preparation  sufficiently  to  coagulate  the  albu- 
m  -n.  in  order  that  it  may  not  be  washed  off  in  the  subsequent  stain- 
ing process.  This  is  best  done,  in  accordance  with  Koch's  directions 
for  the  preparation  of  tuberculous  sputum,  by  passing  the  cover 
glass,  held  in  slender  forceps,  rather  quickly  through  the  flame  of  an 
alcohol  lamp  three  times  in  succession.  In  this  operation  it  must 
ba  remembered  that  too  much  heat  will  destroy  the  preparation, 
while  too  little  will  fail  to  accomplish  the  object  in  view — coagu- 
lation of  the  albumen.  In  passing  the  cover  glass  through  the 
thine  the  smeared  side  is  to  be  held  upward.  The  time  required 
will  be  about  three  seconds  for  passing  it  three  times  as  directed ; 
but  this  will  vary  according  to  the  intensity  of  the  flame,  and  some 
little  experience  is  necessary  in  order  to  obtain  the  best  results. 

The  operation  of  "  fixing,"  or  coagulating  the  albumen,  may  also 
be  effected  by  exposure  in  a  dry-air  oven,  heated  to  120°  to  130°  C., 
for  a  few  minutes  (two  to  ten  minutes),  as  directed  by  Ehrlich. 

Bacteria  simply  suspended  in  distilled  water  adhere  very  well  to 
the  cover  glass  when  treated  as  directed,  but  if  they  have  been  taken 
from  a  liquefied  gelatin  culture  the  film  is  very  apt  to  be  washed 
away  during  the  staining  process.  This  is  best  avoided  by  taking  as 
little  as  possible  of  the  gelatin  medium  and  suspending  the  bacteria 
to  be  examined  in  a  drop  of  water,  which  dilutes  the  gelatin  and 
washes  it  away  from  the  surface  of  the  cells. 

Smear  Preparations. — In  various  infectious  diseases  bacteria  are 
found  in  the  blood  and  tissues  of  the  body,  and  their  presence  may 
be  demonstrated  by  making  what  is  called  a  smear  preparation.  A 
little  drop  of  blood  may  be  spread  upon  the  thin  glass  cover,  or  it 
may  be  brought  in  contact  with  the  freshly  cut  surface  of  one  of  the 
vascular  organs,  as  the  liver  or  spleen.  It  is  especially  desirable  that 
the  material  used  for  such  a  preparation  be  small  in  amount  and  dis- 
tributed evenly  in  a  very  thin  layer.  In  Germany  it  is  the  custom, 
in  making  smear  preparations,  to  press  the  material  between  two  glass 
covers,  which  are  then  separated  by  sliding  them  apart,  thus  leaving 
a  thin  layer  upon  each.  This  answers  very  well,  but  the  writer  pre- 
fers to  spread  the  material  by  drawing  across  the  face  of  the  cover 
glass  the  end  of  a  well-ground  and  polished  glass  slide.  This  method 
i-  « 'specially  useful  for  spreading  blood  in  a  uniform  layer,  in  which 
the  corpuscles  are  evenly  distributed  and  retain  their  normal  form. 
A  very  small  drop  of  blood  is  placed  near  one  edge  of  the  cover  glass, 
which  is  placed  u]x>n  a  smooth  surface  ;  the  glass  slide  is  held  at  a 
very  acute  angle  and  is  gently  drawn  across  the  cover  glass,  without 
any  pressure. 

Most  bacteriologists  make  their  preparations  upon  tlu»  covor 'glass. 


STAINING   METHODS.  27 

as  above  described,  but  the  writer  has  for  a  number  of  years  made 
his  mounts  of  bacteria  upon  the  glass  slide,  and  believes  that  this 
method  has  some  advantages  for  every-day  work.  The  thin  glass 
covers  required  when  a  preparation  is  to  be  examined  with  an  im- 
mersion objective  of  high  power,  are  easily  broken  and  often  dropped 
from  the  fingers  or  forceps.  When  the  material  to  be  examined  is 
spread  and  dried  directly  upon  the  glass  slide,  the  operation  is  at- 
tended with  less  difficulty  and  fewer  accidents  and  the  results  are 
quite  as  good.  In  this  case  the  slide  is  held  in  the  fingers  during  the 
various  steps  in  the  operation  of  distributing,  drying,  and  staining, 
while  the  thin  glass  cover  must  be  held  in  delicate  forceps. 

Contact  Preparations. — When  a  dry  and  clean  cover  glass  is 
brought  in  contact  with  a  colony  or  surface  culture  we  may  often 
obtain  a  very  pretty  preparation,  showing  the  bacteria  in  a  single 
layer,  and  preserving  the  arrangement,  as  regards  growth,  which 
characterizes  the  species.  Similar  preparations  may  sometimes  be 
obtained  from  the  surface  of  liquid  cultures,  when  the  bacteria  grow 
upon  the  surface  as  a  thin  film.  The  cover  glass  is  to  be  gently 
brought  into  contact  with  this  surface  growth,  which  adheres  to  it 
and  is  dried  and  stained  by  the  usual  methods. 

Stain  ing  of  the  dried  film  is  quickly  effected  by  using  an  aqueous 
solution  of  one  of  the  aniline  colors  above  mentioned.  For  general 
use  the  writer  prefers  a  solution  of  f  uchsin,  on  account  of  the  prompt- 
ness of  its  staining  action,  and  because,  in  preparations  for  permanent 
preservation,  it  is  not  as  likely  to  fade  as  methylene  blue  or  gentian 
violet.  It  is  also  a  better  color  than  blue  or  violet  in  case  a  photo- 
micrograph is  to  be  made  from  the  preparation. 

It  is  best  to  keep  on  hand  saturated  alcoholic  solutions  of  the 
staining  agents  named,  and  to  make  an  aqueous  solution  whenever 
required  by  the  addition  of  a  few  drops  to  a  little  water  in  a  watch 
glass  or  test  tube  ;  for  the  aqueous  solutions  do  not  keep  well  on  ac- 
count of  the  precipitation  of  the  dye  as  a  fine  powder,  which  ren- 
ders the  solution  opaque.  The  addition  of  ten  per  cent  of  alcohol 
to  the  aqueous  solution  will,  however,  prevent  this  precipitation ; 
but,  as  a  rule,  freshly  prepared  solutions  are  the  best.  These  should 
be  filtered  before  use.  We  may  place  a  few  drops  of  the  filtered 
solution  upon  the  dried  film  on  the  slide  or  cover  glass,  or  the  thin 
cover  may  be  floated  upon  a  little  of  the  solution  in  a  watch  glass. 
In  some  cases  it  is  best  to  use  heat  to  expedite  the  staining,  and  this 
may  be  done  by  holding  the  slide  or  the  watch  glass  over  the  flame 
of  an  alcohol  lamp  until  steam  commences  to  be  given  off.  If  the 
heating  is  carried  too  far  the  preparation  is  likely  to  be  spoiled  by 
the  precipitation  of  the  staining  agent.  As  a  rule,  heating  will  not 
be  necessary,  and  when  an  aqueous  solution  of  fuchsin  (one  part  to 


28  STAINING  METHODS. 

one  hundred  of  water)  is  used  most  bacteria  are  stained  within  a 
few  seconds  to  a  minute.  At  the  end  of  this  time  the  staining  solu- 
tion is  to  be  washed  away  by  means  of  a  gentle  stream  of  water,  or 
by  moving  the  cover  glass  about  in  a  vessel  containing  distilled 

water. 

Decolorization. — It  often  happens  that  the  albuminous  material 
associated  with  the  bacteria  which  we  propose  to  examine  is  stained 
so  deeply  as  to  obscure  the  view  of  these  ;  and,  generally,  we  will 
obtain  more  satisfactory  preparations  by  the  use  of  a  decolorizing 
agent,  by  which  the  background  is  cleared  up  and  the  outlines  of  the 
cells  more  clearly  defined.  The  agents  chiefly  used  for  this  purpose 
are  alcohol,  diluted  acids,  and  solution  of  iodine  with  potassium 
iodide  (Gram's  solution). 

Koch  recommends  a  solution  containing  sixty  parts  of  alcohol  to 
forty  parts  of  water.  The  cover  glass  is  to  be  quickly  passed 
through  this  solution  two  or  three  times.  Some  bacteriologists  pre- 
fer to  use  absolute  alcohol. 

Or  we  may  use  dilute  acetic  acid  (one-half  to  one  per  cent)  or 
very  dilute  hydrochloric  acid  (ten  drops  to  half  a  litre  of  water). 

For  decolorizing  preparations  containing  the  tubercle  bacillus 
strong  solutions  of  the  mineral  acids  are  employed  (one  part  of  ni- 
tric or  of  sulphuric  acid  to  three  parts  of  water). 

Gram's  solution  contains  one  part  of  iodine  and  two  parts  of 
potassic  iodide  in  three  hundred  parts  of  water.  Special  directions 
will  be  given  for  the  use  of  these  agents  when  we  give  an  account 
of  the  staining  methods  most  useful  for  the  various  pathogenic 
organisms. 

Double  Staining. — After  decolorizing  the  background  of  albu- 
minous material  we  may  again  stain  this  with  a  contrast  stain, 
such  as  eosin  or  vesuvin.  In  mounts  made  from  pure  cultures, 
either  liquid  or  solid,  a  single  stain,  for  the  bacteria  only,  is  all  that 
we  require,  and  our  aim  is  to  have  the  background  as  free  as  possi- 
ble from  any  material  which  would  obscure  the  view. 

After  staining,  decolorizing,  and  washing  the  preparation  the 
cover  glass  or  slide  is  again  dried  by  exposure  to  the  air  or  gentle 
heat,  and  is  then  ready  for  the  permanent  mounting  in  Canada  bal- 
sam. If  the  bacteria  have  been  stained  upon  the  slide,  a  small  drop 
of  balsam  dissolved  in  xylol  is  placed  in  the  middle  of  the  prepara- 
tion and  a  clean,  thin  glass  cover  applied. 

If  it  is  the  intention  to  make  the  microscopical  examination  with 
an  immersion  objective  of  high  power,  or  to  make  photomicro- 
graphs from  it,  only  the  thinnest  glass  covers  should  be  used — one- 
two-hundredths  of  an  inch  or  less. 

If  the  preparation  is  not  intended  for  permanent  preservation, 


STAINING   METHODS.  29 

the  examination  may  be  made  without  drying  the  surface  upon 
which  the  stained  bacteria  are  spread,  the  water  taking  the  place  of 
balsam  in  a  permanent  mount ;  or  we  may  dry  the  film  and  use  a 
drop  of  cedar  oil  between  the  slide  and  cover. 

While  simple  aqueous  solutions  of  the  aniline  colors,  when 
freshly  prepared,  will  promptly  stain  most  bacteria,  certain  agents 
may  be  added  to  these  which  aid  in  the  preservation  of  the  solution, 
or  which  act  as  mordants,  and  are  useful  in  special  cases. 

We  shall  only  give  here  a  few  of  the  standard  solutions  which 
are  most  frequently  employed  by  experienced  bacteriologists  : 

1.  Aniline-Gentian-Violet  (Ehrlich). 

*  Saturated  alcoholic  solution  of  gentian  violet,         .          .  5  cc. 

Aniline  water,       .          .          .          .          .          .          .  100  cc. 

2.  Aniline-Methyl-Violet  (Ehrlich- Weigert). 

Saturated  alcoholic  solution  of  methyl  violet,          .          .          11  cc. 
Absolute  alcohol,  ......  10  cc. 

Aniline  water,  .......         100  cc. 

Aniline  water  for  the  above  solutions  is  prepared  by  shaking  in  a 
test  tube  one  part  of  aniline  oil  with  twenty  parts  of  distilled  water, 
and,  after  allowing  it  to  stand  for  a  short  time,  filtering  the  saturated 
aqueous  solution  through  a  moistened  filter.  If  the  solution  is  not 
perfectly  transparent  it  should  be  filtered  a  second  time. 

3.   Carbol-Fuchsin  (ZiehPs  solution). 

Fuchsin,         .........      1  gm. 

Alcohol,  10  cc. 

Dissolve  and  add  100  cc.  of  a  five-per-cent  solution  of  carbolic  acid. 

4.  Alkaline  Blue  Solution  (Loffler's  solution). 

Saturated  solution  of  methyl ene  blue,        ...  30  cc. 

Solution  of  caustic  potash  of  1 : 10, 000,  .  100  cc. 

These  solutions  keep  better  than  the  simple  aqueous  solutions, 
but  after  having  been  kept  for  a  time  they  are  likely  to  lose  their 
staining  power  as  a  result  of  the  precipitation  of  the  aniline  color. 

The  following  special  methods  of  staining  cover-glass  prepara- 
tions will  be  found  useful  in  certain  cases: 

Gram's  Method.— The  dried  film  upon  a  slide  or  coyer  glass  is 
stained  with  an  aqueous  solution  of  methyl  violet  or  with  aniline- 
gentian-violet  solution  (No.  1);  it  is  then  placed  in  the  iodine  solution 
for  a  minute  or  two  (iodine  one  part,  potassic  iodide  two  parts,  water 


30  STAINING    METHODS. 

thrtM»  hundred  parts);  then  washed  in  alcohol,  dried,  and,  if  for  per- 
manent preservation,  mounted  in  balsam. 

METHODS  OP  STAINING  THE  TUBERCLE  BACILLUS. — Numerous 
methods  of  staining  the  tubercle  bacillus  in  sputum  dried  upon  a 
cover  glass  have  been  proposed,  but  we  shall  only  give  here  two  or 
three  of  the  most  approved  methods,  either  one  of  which  may  be 
relied  upon  for  satisfactory  results  if  carefully  followed. 

1.  The  Ehrlich-  Weigert  Method. — Place  in  a  watch  glass  a  little 
of  the  aniline-methyl-violet  solution  (No.   2);  float  upon  the  surface 
of  this  the  cover  glass  with  the  dried  film  downward  ;  heat  over  a 
small  flame  until  it  begins  to  steam,  then  allow  it  to  stand  for  from 
two  to  five  minutes  ;  decolorize  in  a  tray  cont  lining  one  part  of  nitric 
acid  to  three  parts  of  water— the  cover  glass,  held  in  forceps,  is  gently 
moved  about  in  the  decolorizing  solution  for  a  few  seconds.     It  is 
then  washed  off  in  sixty-per-cent  alcohol  to  remove  the  remaining 
blue  color — this  usually  takes  but  a  second  or  two — and  then  in  water. 
For  a  contrast  stain  a  saturated  aqueous  solution  of  vesuvin  may  be 
used,  a  few  drops  being  left  upon  the  cover  glass  for  five  minutes. 
The  stained   preparation  is  then  washed,  dried,   and  mounted  in 
balsam. 

2.  The  Ziehl-Neelson  Method. — Float  the  cover  glass  upon  the 
carbol-fuchsin  solution  (No.  3) ;  heat  gently  until  steam  commences 
to  rise — from  three  to  five  minutes'  time  will  usually  be  sufficient ; 
wash  off  in  water,  and  decolorize  in  nitric  or  sulphuric  acid,  twenty- 
five-per-cent  solution,  then  in  sixty-per-cent  alcohol  for  a  very  short 
time  to  remove  remaining  color  from  albuminous  background;  wash 
well  in  water  and  mount  in  Canada  balsam. 

3.  Friedlander's  Method. — Spread  and  dry  the  sputum  upon 
the  slide ;  fix  by  passing  the  slide  three  times  through  the  flame  of 
an  alcohol  lamp  or  Bunsen  burner  ;  place  upon  the  dried  film  three  or 
four  drops  of  carbol-fuchsin  (No.  3);  heat  gently  over  a  flame  until 
steam  is  given  off  ;  wash  in  a  dish  of  distilled  water  ;  drain  off  excess 
of  water,  and  add  a  few  drops  of  the  following  decolorizing  solution : 

Acid,  nitric,  pure,         .  .  .  .  5  cc. 

Alcohol  (eighty  per  cent),  .  .  .  .to  100  cc. 

—usually  the  preparation  will  be  decolorized  in  about  half  a  minute  ; 
wash  in  water  ;  add  a  few  drops  of  an  aqueous  solution  of  methylene 
blue  a-  ;i  contrast  stain  ;  allow  the  stain  to  act  for  about  five  minutes, 
without  heating ;  wash  again  in  water,  dry,  and  mount  in  balsam, 
or  for  a  temporary  mount  use  a  drop  of  cedar  oil. 

1.  <;<ihh<'trtt  Method—  This  is  a  slight  modification  only  of  a 
very  useful  method  recommended  by  H.  Frankel  in  1884.  The  con- 
trast stain  is  a«lde«l  to  tin-  decolorizing  solution.  After  staining  with 


STAINING   METHODS.  31 

carbol-fuchsin  solution  (No.  3)  the  cover  glass  is  placed  for  one  or 
two  minutes  in  a  solution  containing: 

Sulphuric  acid  (tvventy-five-per-cent  solution),         .  .       100  cc. 

Methylene  blue,      ......  2  gms. 

Wash,  dry,  and  mount  in  cedar  oil  or  balsam. 

METHODS  OF  STAINING  SPORES. — When  preparations  containing 
the  spores  of  bacilli  are  stained  by  any  of  the  methods  above  given, 
these  remain  unstained  and  appear  as  highly  refractive  bodies  in  the 
interior  of  the  rods  or  filaments  in  which  they  have  been  formed,  or 
scattered  about  in  the  field  if  they  have  been  set  free.  Owing  to 
the  contrast  with  the  stained  protoplasm  of  the  rod  or  spore-bearing 
filament,  they  are  especially  well  seen  in  recent  cultures ;  while  in 
older  cultures  the  bacilli  often  do  not  stain  well,  or  are  entirely  dis- 
integrated and  spores  only  are  to  be  seen.  The  discovery  was  made 
at  about  the  same  time  by  Buchner  (1884)  and  by  Hueppe  that 
spores  may  be  stained  if  they  are  first  exposed  to  an  elevated  tem- 
perature for  some  time.  This  may  be  accomplished  by  placing  the 
slide  or  cover  glass,  upon  which  the  spore-containing  culture  has 
been  dried,  in  a  hot-air  oven  at  a  temperature  of  120°  C.  for  an 
hour;  or  a  higher  temperature  (180°  C.)  may  be  employed  for  a 
shorter  time  (fifteen  minutes)  ;  or  the  cover  glass  may  be  passed 
through  the  flame  of  an  alcohol  lamp  or  Btmsen  burner  eight  or  ten 
times,  instead  of  three  times  as  is  customary  when  the  object  in 
view  is  simply  to  coagulate  the  albumen  and  fix  the  film  upon  the 
cover  glass.  After  such  treatment  the  spores  may  be  stained  with 
an  aqueous  solution  of  one  of  the  basic  aniline  colors — fuchsin, 
methyl  violet,  etc. — but  the  bacilli  no  longer  take  the  stain  so  well. 

To  obtain  satisfactory  double-stained  preparations,  showing 
both  spores  and  bacilli,  a  different  method  is  employed. 

The  film  upon  the  cover  glass  is  passed  through  the  flame  three 
times,  as  heretofore  directed  ;  it  is  then  floated  upon  aniline-f uchsin 
solution  in  a  watch  glass,  and  this  is  heated  to  near  the  boiling  point 
for  an  hour — Neisser's  method.  The  aniline-fuchsin  solution  is 
prepared  by  shaking  an  excess  of  aniline  oil  in  a  test  tube  with  dis- 
tilled water,  filtering  the  saturated  solution  into  a  watch  glass,  and 
then  adding  a  few  drops  of  a  saturated  alcoholic  solution  of  fuchsin. 
After  this  prolonged  action  of  the  hot  staining  fluid  the  spores  of 
some  bacilli  are  deeply  stained,  while  others  do  not  take  the  stain  so 
well.  The  cover  glass  is  next  washed  in  water  and  then  placed  in 
a  decolorizing  solution  containing  twenty-five  parts  of  hydrochloric 
acid  to  seventy-five  parts  of  alcohol.  This  removes  the  stain  from 
the  bacilli,  but,  if  not  allowed  to  act  too  long,  leaves  the  spores  still 
stained.  The  preparation  is  next  stained  in  a  saturated  aqueous 


32  STAINING  METHODS. 

solution  of  methylene  blue;  and  if  the  operation  has  been  successfully 
carried  out  the  spores  will  be  stained  red  and  the  protoplasm  of  the 
bacilli  in  which  they  are  present  will  be  blue. 

Moller  has  (1891)  published  the  following  method  of  staining 

spores : 

The  cover-glass  preparation,  dried  in  the  air,  is  passed  three  times 
through  a  flame  or  placed  for  two  minutes  in  absolute  alcohol ;  it  is 
then  placed  in  chloroform  for  two  minutes  and  washed  in  water;  it 
is  now  immersed  in  a  five-per-cent  solution  of  chromic  acid  for  from 
half  a  minute  to  two  minutes  and  again  thoroughly  washed  in 
water;  next  a  solution  of  carbol-fuchsin  is  poured  upon  it  and  it 
is  heated  over  a  flame  until  it  commences  to  boil,  for  sixty  seconds ; 
the  carbol-fuchsin  solution  is  then  poured  off  and  the  cover  glass  is 
immersed  in  a  five-per-cent  solution  of  sulphuric  acid  until  the 
film  is  decolorized,  after  which  it  is  again  thoroughly  washed  in 
water.  It  is  then  placed  for  thirty  seconds  in  an  aqueous  solution  of 
methylene  blue  or  of  malachite  green,  and  again  washed  in  water, 
after  which  the  preparation  should  be  dried  and  mounted  in  balsam. 
As  a  result  of  this  procedure  the  spores  are  stained  dark  red  and  the 
protoplasm  of  the  bacilli  blue  or  green. 

Fiocca  (18915)  claims  that  better  results  are  obtained  by  the  follow- 
ing method : 

About  twenty  cc.  of  a  ten-per-cent  ammonia  solution  is  placed  in  a 
watch  glass,  and  from  ten  to  twenty  drops  of  an  alkaline  solution  of 
an  aniline  color  is  added ;  heat  is  applied  until  steam  commences  to  be 
given  off,  when  the  cover  glass  is  placed  in  the  hot  solution  for  from 
three  to  fifteen  minutes.  The  cover  glass  is  then  quickly  washed  in 
a  twenty-per-cent  solution  of  nitric  or  sulphuric  acid  to  decolorize ; 
then  it  should  be  thoroughly  washed  in  water,  after  which  it  may 
be  stained  with  a  contrast  color  by  the  use  of  an  aqueous  solution  of 
one  of  the  aniline  dyes — preferably  vesuvin,  malachite  green,  or 
safranin. 

METHODS  OF  STAINING  FLAGELLA.— Koch  first  succeeded  in  de- 
monstrating the  flagella  of  certain  bacilli  and  spirilla  by  staining 
them  with  an  aqueous  solution  of  ha3inatoxylon,  and  dilute  chromic 
acid  as  a  mordant.  Loflfler  (1889)  has  succeeded  in  demonstrating, 
by  an  improved  staining  method,  the  presence  of  flagella  in  a  consider- 
able number  of  species  in  which  they  had  not  previously  been  seen, 
although  generally  suspected  to  be  present.  His  method  is  as  follows : 

Loffler's  Method.— The  following  solution  is  used  as  a  mordant: 

No.  1. 

Solution  of  tannin  of  twenty  per  cent,          .  .10  cc. 

Saturated  (coldi  solution  of  ferrous  sulphate,    .  .  .     5  cc. 

Aqueous  or  alcoholic  solution  of  fuchsin,     .  .  1  cc. 

((  >r  one  cubic  centimetre  alcoholic  solution  of  methyl  violet.) 


STAINING   METHODS.  33 

No.  2. 

A  one-per-cent  solution  of  caustic  soda. 

No.  3. 

A  solution  of  sulphuric  acid  of  such  strength  that  one  cubic  centimetre 
is  exactly  neutralized  by  one  cubic  centimetre  of  the  soda  solution. 

According  to  Loffler,  solution  No.  1  is  just  right  for  staining  the 
flagellum  of  Spirillum  concentricum,  but  for  certain  other  bacteria  it 
is  necessary  to  add  to  this  some  of  No.  2  or  of  No.  3.  Thus,  for  the 
cholera  spirillum  from  half  a  drop  to  a  drop  of  the  acid  solution  is 
added  to  sixteen  cubic  centimetres  of  No.  1.  For  the  bacillus  of 
typhoid  fever  one  cubic  centimetre  of  No.  2  is  added  to  sixteen  cubic 
centimetres  of  No.  1.  Bacillus  subtilis  requires  twenty-eight  to 
thirty  drops  of  No.  2 ;  the  bacillus  of  malignant  oedema  thirty-six  to 
thirty-seven  drops,  etc. 

This  method  has  not  been  very  successful  in  the  hands  of  other 
bacteriologists,  and  improvements  in  the  technique  have  been  made 
since  it  was  first  published.  Van  Ermengem  (1893)  points  out  the 
fact  that. a  principal  condition  of  success  is  that  the  cover  glasses  shall 
be  absolutely  clean.  He  boils  them  in  a  mixture  composed  of  potas- 
sium bichromate,  sixty  grammes;  concentrated  sulphuric  acid,  sixty 
grammes ;  water,  one  hundred  grammes.  After  coming  from  this  they 
are  thoroughly  washed  in  water,  then  in  absolute  alcohol,  and  then 
dried  in  an  upright  position  under  a  bell-jar.  Recent  agar  cultures 
(ten  to  eighteen  hours)  are  preferred,  and  the  suspension  in  water 
should  be  very  much  diluted  so  that  in  the  cover-glass  preparation 
the  bacteria  are  well  isolated.  The  cover  glass,  held  between  the 
fingera,  is  passed  three  times  through  a  flame.  A  drop  of  the  follow- 
ing solution  is  then  placed  upon  it:  Osmic  acid  two-per-cent  solution, 
one  part ;  solution  of  tannin  (ten  to  twenty-five  per  cent)  two  parts. 
This  is  allowed  to  act  for  about  five  minutes  at  a  temperature  of  50° 
to  60°  C. — or  half  an  hour  at  the  room  temperature.  After  careful 
washing  with  water  and  alcohol  the  cover  glass  is  immersed  for  a 
few  seconds  in  a  bath  containing  one-quarter  to  one-half  per  cent  of 
nitrate  of  silver.  Then  without  washing  it  is  placed  for  a  short 
time  in  the  following:  Gallic  acid,  five  grammes;  tannin,  three 
grammes;  fused  potassium  acetate,  ten  grammes;  distilled  water, 
three  hundred  and  fifty  grammes.  It  is  then  returned  to  the  silver 
bath  and  kept  there,  with  constant  movement  of  the  bath,  until  this 
commences  to  turn  black.  It  is  then  thoroughly  washed  in  water, 
dried,  and  mounted  in  balsam. 

Pitfield  (1895)  has  devised  a  much  simpler  method  which  he  de- 
scribes as  follows : 

"The  method  consists  in  the  use  of  but  a  single  solution,  which  is  at  once 
mordant  and  stain.     The  solution  should  be  made  in  two  parts,  which  are 
filtered  and  mixed. 
3 


34  STAINING  METHODS. 


Saturated  aqueous  solution  of  alum,         .  .10  c.c. 

Saturated  alcoholic  solution  of  gentian -violet,          .  .    1  c.c. 

B. 

Tannicacid,  .....-•          1  g™- 
Distilled  water, 10  c.c. 

"The  solutions  should  be  made  with  cold  water,  and  immediately  after 
mixing  the  stain  is  ready  for  use, 

"  The  cover  slip  is  to  be  carefully  cleaned,  the  grease  being  burned  off  m  a 
flame,  and  after  it  has  cooled  the  bacteria  are  spread  upon  it,  well  diluted  in 
water,  care  being  taken  to  exclude  culture  medium.  After  the  preparation 
has  been  thoroughly  dried  in  the  air  it  should  be  held  over  the  name  with  the 
fingers  (the  preparation  need  not  be  fixed)  as  Loftier  has  directed.  After- 
ward the  stain  is  gradually  poured  on  the  slip  and  heated  gently,  bringing 
the  fluid  almost  to  a  boil ;  the  slip  covered  with  the  hot  stain  should  then  be 
laid  aside  for  one  minute,  then  washed  in  water  and  mounted. 

**  If  the  filtered  stain  is  used,  a  second  stain  of  aniline  water  containing 
gentian-violet  had  better  be  used,  which  should  be  applied  but  a  moment  and 
thru  washed  off,  thus  leaving  a  clean  field,  showing  only  bacteria  lightly 
stained,  with  their  flagella  still  more  lightly  colored." 

METHODS  OF  STAINING  BACTERIA  IN  TISSUES. — The  solutions  re- 
commended for  staining  cover-glass  preparations  are  also  used  in 
staining  bacteria  in  thin  sections  of  the  various  organs,  in  which 
they  are  found  in  certain  infectious  diseases;  but,  in  general,  a 
longer  time  is  required  to  stain  sections,  and  it  is  best  not  to  hasten 
the  process  by  the  use  of  heat.  To  obtain  good  thin  sections,  the 
material,  cut  in  small  cubes,  must  be  very  thoroughly  hardened  in 
absolute  alcohol.  The  piece  selected  for  cutting  may  be  attached  to 
a  cork  by  the  use  of  melted  glycerin  jelly,  which  is  hardened  by 
placing  the  cork  and  attached  piece  of  tissue  in  alcohol.  This  an- 
swers for  well-hardened  pieces  of  liver,  kidney,  etc. ,  but  the  hollow 
viscera  and  tissues  of  loose  structure  will  require  embedding  in 
paraffin  or  celloidin.  Any  well-made  sledge  microtome  will  answer 
t  •  >r  cutting  the  sections,  if  the  knife  is  properly  sharpened.  The  sec- 
tions should,  of  course,  be  cut  under  alcohol,  and  they  can  scarcely 
be  too  thin  when  the  object  is  to  demonstrate  the  presence  or  ab- 
sence of  bacteria.  Very  thin  sections  may  be  cut  dry  by  embedding 
in  paraffin  having  a  melting  point  of  50°  C.  In  this  case  the  knife 
is  set  at  a  right  angle  to  the  material  to  be  cut,  and  the  sections 
are  spread  out  upon  and  attached  to  the  glass  slide  for  staining. 

One  of  the  most  useful  solutions  for  staining  tissues  is  Loffler's 
alkaline  solution  of  methylene  blue  (No.  4).  A  freshly-prepared  so- 
lution \\ill  stain  sections  in  four  or  five  minutes.  Superfluous  color 
is  removed  by  immersing  the  sections  in  diluted  alcohol  or  in  a  one- 
half-|K'r-(vnt  solution  of  acetic  acid  for  a  few  seconds.  The  sections 


STAINING  METHODS.  35 

are  dehydrated  in  absolute  alcohol,  cleared  up  with  oil  of  cedar,  and 
mounted  in  a  drop  of  cedar  oil  for  examination,  or  in  balsam  if 
they  are  to  be  preserved. 

Gram's  method  may  be  used  as  directed  for  cover-glass  prepara- 
tions, the  sections  being  first  stained  in  aniline-gentian-violet  solu- 
tion (No.  1),  then  washed  in  water,  or  in  aniline  water  as  recently 
(1892)  recommended  by  Botkin,  then  decolorized  in  the  iodine  solu- 
tion (see  page  29).  The  sections  when  decolorized  are  again  washed 
in  water,  dehydrated  in  absolute  alcohol,  cleared  in  cedar  oil,  and 
mounted  in  balsam. 

Weigert's  Method. — This  is  a  modification  of  Gram's  method  in 
which  the  sections  are  dehydrated  by  the  use  of  aniline  oil.  The 
stained  section,  after  having  been  washed,  is  transferred  to  a  clean 
glass  slide,  the  excess  of  water  is  removed  by  the  use  of  filtering 
paper,  and  the  iodine  solution  is  placed  upon  it  in  sufficient  quantity 
to  cover  the  entire  section.  When  sufficiently'  decolorized  this  is  re- 
moved in  the  same  way.  The  section  is  then  dehydrated  by  placing 
a  few  drops  of  aniline  oil  upon  it,  removing  this  with  filtering  paper, 
and  repeating  the  operation  once  or  twice.  The  aniline  oil  must 
then  be  completely  removed  by  the  use  of  xylol,  after  which  the  sec- 
tion is  mounted  in  balsam. 

Kuhne's  Method.— The  object  of  this  method  is  to  prevent  the  removal 
of  the  color  from  stained  bacteria  in  sections  during  the  treatment  which 
such  sections  usually  receive  before  they  are  ready  for  mounting — i.e., 
during  the  washing  and  dehydrating  processes  usually  employed.  For 
staining,  Kiihne  prefers  a  methylene-blue  solution  prepared  as  follows: 
Methylene  blue,  1.5  parts;  absolute  alcohol,  ten  parts;  triturate  in  a  watch 
glass  and  add  gradually  one  hundred  parts  of  a  solution  of  carbolic  acid 
containing  five  parts  in  one  hundred  of  water.  The  section  is  placed  in  this 
solution  for  about  half  an  hour,  then  washed  in  water  and  decolorized  in  a 
weak  solution  of  hydrochloric  acid — ten  drops  to  five  hundred  grammes  of 
water.  This  part  of  the  operation  must  be  conducted  very  carefully,  and 
usually  thin  sections  will  only  require  to  be  dipped  in  the  acid  solution  for  an 
instant,  after  which  they  must  be  at  once  immersed  in  a  solution  of  lithium 
— eight  drops  of  a  saturated  solution  of  carbonate  of  lithium  in  ten  grammes 
of  water.  They  are  then  allowed  to  remain  in  a  bath  of  distilled  water  for 
a  few  minutes,  after  which  they  are  dipped  into  absolute  alcohol,  which 
Kiihne  colors  by  the  addition  of  methylene  blue.  The  sections  are  then 
placed  in  aniline  oil  which  contains  a  little  methylene  blue  in  solution, 
where  they  are  dehydrated  without  the  color  being  extracted  from  the  stained 
bacteria  present.  The  aniline-oil  blue  solution  is  prepared  by  adding  an  ex- 
cess of  dry  methylene  blue  to  a  small  quantity  of  clarified  aniline  oil.  The 
undissolved  pigment  settles  to  the  bottom,  and  a  few  drops  of  the  colored 
solution  are  added  to  a  little  aniline  oil  in  a  watch  glass  to  make  the  colored 
dehydrating  bath.  The  section  is  next  washed  out  in  pure  aniline  oil— not 
colored — after  which  every  trace  of  aniline  oil  is  to  be  removed  by  the  use 
*  xylol.  The  section  is  cleared  up  in  turpentine  and  mounted  in  balsam. 

Ziehl-Neelson  Method,  for  the  tubercle  bacillus  in  tissues. — 
Leave  the  sections  for  fifteen  minutes  in  carbol-fuchsin  solution 
(No.  3) ;  decolorize  in  sulphuric  or  nitric  acid,  twenty-five-per-cent 


36  STAINING   METHODS. 

solution;  wash  in  sixty-per-cent  alcohol;  place  in  a  saturated  aque- 
ous solution  of  methylene  blue  for  contrast  stain;  wash,  dehydrate, 
and  mount  in  balsam. 

The  following1  method  of  staining  sections  for  the  purpose  of  demon- 
strating bacteria  present  in  the  tissues  is  recommended  by  Pregl  (1891)  as  a 
substitute  for  the  method  of  Kuhne.  The  results  are  said  to  be  excellent, 
and  it  is  much  simpler  and  more  expeditious. 

The  sections  are  made  from  tissues  embedded  in  paraffin,  and  are  attached 
to  clean  glass  slides  with  albumen-glycerin.  Or  they  may  be  attached  to  a 
cover  glass  by  the  following  method  when  not  embedded  in  paraffin :  The 
sections,  completely  dehydrated,  are  taken  out  of  absolute  alcohol  on  a  thin 
glass  cover,  upon  which  they  are  extended  ;  a  piece  of  filter  paper  is  applied 
to  the  side  of  the  cover  glass  to  absorb  the  alcohol,  and  before  the  section  is 
completely  dry  a  drop  of  aceton-celloidin  solution  is  placed  upon  it  by  means 
of  a  glass  rod.  The  cover  glass  is  now  moved  about  in  the  air  to  promote 
rapid  evaporation  of  the  alcohol,  and  is  then  placed  in  water.  The  section 
now  remains  attached  to  the  cover  glass  during  subsequent  manipulations. 
The  aceton-celloidin  solution  referred  to  is  prepared  by  adding  celloidin  in 
small,  dry  pieces  to  aceton  until  a  concentrated  solution  is  obtained.  A 
large  drop  of  this  added*to  five  cubic  centimetres  of  absolute  alcohol  makes 
a  suitable  solution  for  use.  This  must  be  kept  in  a  glass-stoppered  bottle,  and 
will  require  to  be  frequently  renewed,  as  it  is  not  suitable  for  use  after  hav- 
ing absorbed  moisture  from  the  air.  The  aceton  as  obtained  from  dealers 
contains  considerable  water  and  must  be  dehydrated  by  adding  to  it  red-hot 
sulphate  of  copper. 

The  sections,  attached  to  a  slide  or  cover  glass  by  one  of  the  methods 
mentioned,  are  stained  with  Kuhne's  carbol-methylene-blue  solution,  which 
is  drooped  upon  them  from  a  pipette.  Usually  they  will  be  sufficiently 
stained  at  the  end  of  half  a  minute  to  a  minute,  but  in  some  cases  a  longer 
time  and  the  application  of  heat  will  be  desirable.  They  are  then  washed  in 
water  and  immediately  placed  in  fifty-per-cent  alcohol,  where  they  remain 
until  the  sections  have  a  Dale-blue  color  with  a  greenish  tinge.  They  are 
now  completely  dehydrated  in  absolute  alcohol  and  subsequently  cleared  up 
in  xylol. 

STAINING  SECTIONS  OF  GELATIN  STICK  CULTURES.— Fischl,  Weigert, 
and  Neisser  have  given  an  account  of  methods  for  staining  stick  cultures  in 
gelatin  of  non-liquefying  bacteria.  The  object  of  this  is  to  show  the  mode 
of  growth  and  the  association  of  individual  cells  in  undisturbed  cultures. 
Neisser  gives  the  following  directions  :  The  gelatin  cultures  are  inoculated, 
by  several  punctures,  with  the  microorganism  to  be  studied.  When  the 
development  is  deemed  sufficient  the  cylinder  of  gelatin  is  removed  from  the 
test  tube  by  gently  warming  its  walls.  It  is  then  placed  for  several  days- 
one  to  eight,  according  to  its  size  and  thickness— in  a  one-per-cent  solution  of 
bichromate  of  potassium.  While  in  this  solution  it  must  be  exposed  to  the 
light,  which  causes  a  change  in  the  gelatin,  rendering  it  insoluble.  The 
gelatin  cylinder  is  thoroughly  washed  and  then  hardened  in  alcohol,  first  of 
seventy  per  cent,  and  then  of  ninety -six  percent.  It  is  then  cut  into  suit- 
able pieces,  and  these  are  attached  to  a  cork  in  the  usual  manner  and  placed 
for  twenty-four  hours  in  absolute  alcohol.  Thin  sections  may  now  be  made 
witli  a  microtome,  and  these  are  attached  to  a  glass  slide  and  stained  by 
<  tram  s  or  Weigerfs  method  or  by  the  use  of  Loffler's  solution  (No.  4).  The 

•olorization  should  he  effected  by  the  use  of  alcohol  and  not  with  an  acid 

ution.  When  Gram's  method  %is  used  decolorize  by  the  alternate  use  of 
alcohol  and  oil  of  cloves.  Clear  the  preparation  with  oil  of  bergamot. 


V. 
CULTURE  MEDIA. 

To  obtain  a  satisfactory  knowledge  of  the  biological  characters 
of  the  different  species  of  bacteria,  it  is  necessary  to  isolate  them  in 
"  pure  cultures  "  and  to  study  their  growth  in  various  culture  media. 
By  a  pure  culture  we  mean  a  cultivation  containing  a  single  species 
only ;  and  to  be  absolutely  sure  that  we  have  a  pure  culture  it  is 
desirable  that  all  of  the  bacteria  in  a  culture  shall  be  the  progeny  of 
a  single  cell.  The  methods  of  obtaining  pure  cultures  will  be  given 
later.  At  present  we  propose  to  give  an  account  of  the  various  cul- 
ture media  commonly  employed  by  bacteriologists,  and  the  methods 
of  preparing  them  for  use. 

By  a  natural  culture  medium  we  mean  one  which,  as  obtained  in 
nature,  contains  the  necessary  pabulum  for  the  development  of  one 
or  more  species  of  bacteria.  An  artificial  culture  medium  is  one 
which  is  prepared  artificially  by  adding  nutritive  material  to  water. 
A  sterile  medium  is  one  which  does  not  contain  any  living  micro- 
organisms. We  may  obtain  natural  media  in  a  sterile  condition,  but 
artificial  media  require  sterilization,  as  they  are  infallibly  contami- 
nated with  living  "  germs  "  from  the  atmosphere  during  the  process 
of  preparing  them.  Sterilization  is  usually  effected  by  heat.  For- 
ceps, glass  tubes,  etc. ,  may  be  sterilized  by  passing  them  through 
the  flame  of  an  alcohol  lamp  or  Bunsen  burner. 

NATURAL  CULTURE  MEDIA. — The  most  important  natural  cul- 
ture medium  is  blood  serum,  which  may  be  obtained  from  one  of 
the  lower  animals — preferably  from  oxen  or  calves.  This  is  to  be 
collected  in  a  sterilized  jar,  with  every  precaution  to  insure  cleanli- 
ness, at  the  moment  of  slaughtering  the  animal.  Or  the  blood  of  a 
calf,  sheep,  or  dog  may  be  collected  at  the  laboratory  by  a  carefully 
conducted  operation,  in  which  the  femoral  or  carotid  artery  is  con- 
nected with  a  sterilized  glass  tube  leading  into  a  sterilized  receptacle; 
such  as  a  Woulf  's  bottle,  into  one  neck  of  which  a  cotton  plug  has 
been  placed  to  permit  the  air  to  escape  as  the  bottle  fills  with 
blood  through  a  tube  which  is  secured  in  the  other  neck.  When 
blood  is  passed  directly  from  an  artery  into  a  sterilized  receptacle 
the  serum  will  not  subsequently  require  sterilization.  The  writer  is  in 


CULTURE   MEDIA. 


the  habit  of  collecting  it  in  this  way,  and,  after  the  serum  has  sepa- 
rated, of  drawing  it  off  in  little  flasks  having  a  long  neck,  as  shown 
in  Fig.  14.  The  neck  of  the  flask,  previously  sterilized  by  heat,  is 
slipped  into  the  Woulf  s  bottle  beside  the  cotton  plug,  the  bulb  (a) 
having  been  previously  gently  heated  to  expand  the  contained  air. 
As  the  heated  air  cools  a  partial  vacuum  is  formed  and  the  clear 
serum  mounts  into  the  little  flask.  One  after  another  is  filled  in 
this  way,  and  each  one  is  hermetically  sealed  in  the  flame  of  a  lamp 


-a 


Fio.  14. 


Fio.  15. 


Fio.  18. 


as  soon  as  it  is  withdrawn.    The  sterile  blood  serum  may  be  pre- 
served indefinitely  in  this  way,  and  may  be  used  as  a  liquid  culture 
me?'U™  -«  *ht  httle  flask'  or  it  may  be  transferred  to  a  test  tube 
and  solidified  by  heat  whenever  a  solid  blood-serum  medium  is  re- 
ft,    $£!*&  °f  PreservinS  bl<*>d  serum  and  other  liquid 
edia  m   hese  httle  flasks  is  in  the  fact  that  they  may  be  preserved 
.definitely  without  becoming  contaminated  or  drying  up,  and  that 
Uiey  are  easily  transported,  while  a  liquid  medium  in  a  test  tube 
must  be  kept  upright.    The  contents  of  one  of  these  flasks  are  readily 


CULTURE   MEDIA.  39 

transferred  to  a  test  tube  by  breaking  off  the  sealed  extremity  with 
sterile  forceps  and  slipping  it  past  the  cotton  plug,  which  must  be 
partly  withdrawn  for  the  purpose.  Upon  applying  gentle  heat  to 
the  bulb  its  contents  are  forced  out  into  the  test  tube  (Fig.  15). 
Blood  serum  which  is  collected  without  these  special  precautions 
will  require  sterilization  by  heat,  for  which  directions  will  be  given 
later. 

To  obtain  the  clear  serum  from  blood  collected  as  above  directed, 
the  jars  containing  it  are  set  aside  in  a  cool  place  in  order  that  a  firm 
clot  may  form,  care  being  taken  not  to  shake  them.  After  the  clot 
has  formed  they  may  be  transported  to  the  laboratory,  where  they 
are  placed  in  an  ice  box  or  in  a  cool  cellar  for  from  twenty-four  to 
forty-eight  hours.  By  this  time  the  serum  has  separated  from  the 
clot,  and  it  may  be  transferred  to  sterilized  test  tubes  by  means  of  a 
suction  pipette  (Fig.  16),  or  may  be  distributed  in  little  flasks  as 
above  directed. 

M ilk  is  largely  used  as  a  culture  medium,  and  is  especially  useful 
in  studying  the  biological  characters  of  various  microorganisms,  as 
shown  by  their  causing  coagulation  of  the  casein,  or  otherwise  ;  or 
an  acid  or  alkaline  reaction  of  the  liquid  ;  or  peptonization  of  the 
precipitated  casein,  etc.  In  the  udder  of  healthy  cows  milk  is  quite 
sterile,  and  by  proper  precautions  it  may  be  drawn  into  sterilized 
flasks  without  any  contamination  and  kept  indefinitely  without  un- 
dergoing coagulation  or  any  other  change.  But  in  practice  it 
is  easier  to  sterilize  it  in  test  tubes  or  small  flasks  by  the  use  of 
heat  than  to  obtain  it  in  a  sterile  condition  from  the  udder  of  the 
cow. 

Urine  has  been  used  to  some  extent  as  a  culture  medium,  and 
many  bacteria  multiply  in  it  abundantly,  although,  on  account  of  its 
acid  reaction,  other  species  fail  to  grow  in  it.  As  contained  in  the 
healthy  bladder  it  is  sterile,  but  the  mucous  membrane  of  the  mea- 
tus  urinarius  always  contains  numerous  bacteria  upon  its  surface,  and 
some  of  these  are  sure  to  be  carried  away  with  the  current  when 
urine  is  passed. 

A  culture  fluid  which  the  writer  has  found  extremely  useful,  in 
tropical  countries  where  it  is  to  be  obtained,  is  the  transparent  fluid 
contained  in  the  interior  of  unripe  cocoanuts — called  agua  coco  by 
the  Spaniards.  In  countries  where  the  cocoanut  is  indigenous  this 
cocoanut  water  is  largely  used  as  a  refreshing  drink.  It  contains 
about  four  per  cent  of  glucose  in  solution,  together  with  some  vege- 
table albumen  and  salts.  Some  microorganisms  multiply  in  it  with- 
out appropriating  the  glucose,  while  others  split  this  up,  producing 
an  abundant  evolution  of  carbon  dioxide  and  giving  to  the  fluid 
a  very  acid  reaction.  The  following  are  the  results  of  an  analysis 


40  CULTURE   MEDIA. 

made  for  me  by  Dr.  L.  L.  Van  Slyke  in  the  chemical  laboratory  of 
Johns  Hopkins  University  :  The  weight  of  the  fluid  obtained  from 
H\  nuts  averaged  339.1  grammes.  The  specific  gravity  averaged 
1.02285.  The  amount  of  water  averaged  95  per  cent ;  the  amount 
of  inorganic  ash,  0.618  per  cent;  the  amount  of  glucose,  3.97  per 
cent ;  the  amount  of  fat,  0.119  per  cent ;  the  amount  of  albuminoids, 
0.133  percent. 

As  this  fluid  is  contained  in  a  germ-proof  receptacle,  no  steriliza- 
tion is  required  when  it  is  drawn  off  with  proper  precautions  in  the 
little  flasks  heretofore  described. 

Hydrocele  fluid  has  been  used  as  a  culture  medium,  and  many 
bacteria  multiply  in  it  abundantly. 

Other  natural  culture  media  are  found  in  the  animal  and  vege- 
table kingdoms,  which  are  used,  either  cooked  or  raw,  as  solid  sub- 
strata upon  which  bacteria  may  be  cultivated.  One  of  the  most  use- 
ful of  these  is  the  potato,  which  is  a  favorable  medium  for  the  de- 
velopment of  numerous  species,  and  upon  which  (cooked)  many  of 
them  present  characters  of  growth  which  are  so  distinctive  as  to  aid 
greatly  in  the  differentiation  of  species. 

Other  tubers,  roots,  or  fruits  may  also  be  used  as  solid  media,  or 
their  juices  extracted  and  employed  as  liquid  media.  Cooked  fish 
and  meats  of  various  kinds  are  also  suitable  media  for  certain  spe- 
cies— e.g.,  the  phosphorescent  bacteria  grow  very  well  upon  the  sur- 
face of  boiled  fish,  and  in  a  dark  room  give  off  a  bright,  phosphores- 
cent light. 

Eggs,  sterilized  by  boiling,  have  been  used  by  some  bacteriolo- 
gists, especially  for  the  cultivation  of  anaerobic  species. 

ARTIFICIAL  CULTURE  MEDIA.— A  great  variety  of  liquid  media 
have  been  employed  by  bacteriologists,  the  most  useful  of  which  are 
infusions  of  beef  or  mutton,  with  the  addition  of  a  little  peptone. 
But  Pasteur  has  shown  that  some  species  of  bacteria  will  grow  in  a 
medium  which  does  not  contain  any  albuminous  material,  nitrogen 
being  obtained  from  salts  containing  ammonia. 

Pasteur's  solution,  which  is  rarely  used  at  present,  contains  : 
Distilled  water,  one  hundred  parts  ;  cane  sugar,  ten  parts ;  tartrate 
of  ammonia,  one  part,  with  the  addition  of  the  ashes  from  one 
gramme  of  yeast. 

Cohn  modified  this  by  leaving  out  the  cane  sugar,  which  favors 
the  development  of  moulds.  These  fluids  are  not,  however,  in- 
tended for  general  use  in  the  cultivation  of  bacteria,  but  to  demon- 
si  rate  certain  facts  relating  to  their  physiology. 

Infusions  of  meat,  or  "  flesh  water,"  are  made  by  chopping  fine 
lean  U-ef  MI-  mutton  (,,11,'  pound)  and  covering  it  with  water  (one 
litre).  This  is  placed  in  an  ice  chest  for  twenty-four  hours,  and  the 


CULTURE   MEDIA.  41 

aqueous  extract  is  then  obtained  by  filtration  through  muslin  by 
pressure.  This  extract  is  cooked,  filtered,  and  carefully  neutralized 
by  the  addition  of  a  solution  of  carbonate  of  sodium,  which  is  added 
drop  by  drop.  Usually  we  add  to  this  one-half  per  cent  of  chloride 
of  sodium.  The  addition  of  ten  grammes  of  peptone  to  a  litre  of 
this  meat  infusion  constitutes  the  flesh-peptone  solution  which  is 
largely  used  in  the  preparation  of  solid  culture  media,  to  be  described 
hereafter. 

The  addition  of  five  per  cent  of  glycerin  to  the  above  infusion 
makes  a  useful  liquid  medium  for  the  cultivation  of  the  tubercle  ba- 
cillus (Roux  and  Nocard).  The  liquid  should  be  again  neutralized 
after  adding  the  glycerin,  which  commonly  has  an  acid  reaction. 

Bouillon  is  made  by  cooking  the  chopped  meat — one  pound  in  a 
litre  of  water — for  about  half  an  hour  in  a  large  glass  flask  or  an 
enamelled  iron  kettle.  The  filtered  bouillon  is  then  carefully  neu- 
tralized with  sodium  carbonate,  and  again  boiled  for  an  hour  to  pre- 
cipitate all  coagulable  albuminoids.  It  is  again  filtered  and  dis- 
tributed in  test  tubes  or  small  flasks,  in  which  it  is  subsequently 
sterilized.  For  certain  pathogenic  bacteria  a  bouillon  made  from  the 
flesh  of  a  fowl  or  of  a  rabbit  is  preferable  to  beef  bouillon. 

Flesh  infusion  may  also  be  made  from  one  of  the  standard  beef 
extracts,  such  as  Liebig's  (five  grammes  to  a  litre  of  water). 

Various  vegetable  infusions  may  also  be  used  as  culture  media, 
such  as  yeast  water,  potato  water,  infusion  of  hay,  of  barley,  or  of 
wheat,  of  dried  fruits,  beer  wort,  etc. 

SOLID  CULTURE  MEDIA. — The  introduction  of  solid  culture 
media,  and  especially  the  use  of  gelatin  and  agar-agar,  as  first 
recommended  by  Koch  (1881),  for  the  isolation  and  differentiation  of 
species,  was  a  most  important  advance  in  bacteriological  technology. 
We  are  concerned  here  only  with  the  composition  and  preparation 
of  these  media. 

Flesh-Peptone-Gelatin. — This  is  made  by  adding  ten  per  cent 
of  the  best  French  gelatin  to  the  flesh-peptone  solution  above  de- 
scribed. This  is  the  standard  gelatin  medium,  but  more  or  less 
gelatin  may  be  added  to  serve  a  special  purpose.  Thus,  in  Havana 
during  the  summer  months  the  writer  used  a  medium  containing 
twenty  per  cent  of  gelatin,  because  when  but  ten  per  cent  was  used 
the  gelatin  was  liquefied  by  the  normal  temperature  of  the  atmo- 
sphere. Teii-per-cent  gelatin,  of  good  quality  and  carefully  pre- 
pared, will  stand  a  temperature  of  20°  to  22°  C.  (68°  to  71. 6°,  F.) 
without  melting.  When  twenty  per  cent  of  gelatin  is  used  the 
melting  point  is  about  8°  C.  higher.  It  must  be  remembered  that 
exposure  to  a  boiling  temperature  reduces  the  melting  point  of  gela- 
tin. It  is  therefore  desirable  to  accomplish  the  operations  of  cook- 


42  CULTURE  MEDIA. 

ing  and  sterilizing  in  as  short  a  time  as  is  practicable.  The  French 
gelatin  used  comes  in  thin  sheets ;  this  is  broken  up  and  added  to 
the  flesh-peptone  solution. 

Usually  we  prepare  a  litre  of  nutrient  gelatin  at  one  time,  and  for 
this  quantity  one  hundred  grammes  of  gelatin  will  be  required  for  the 
standard  preparation  (ten  per  cent).  It  is  well  to  allow  it  to  soak  for 
a  time  in  the  liquid  before  applying  heat  for  the  purpose  of  dissolving 
it.  Then  apply  gentle  heat  until  it  is  completely  dissolved.  The  gela- 
tin of  commerce  usually  has  an  acid  reaction,  and  it  will  be  necessary 
to  carefully  neutralize  the  medium  after  it  has  been  added.  A  slightly 
alkaline  reaction  is  usually  no  disadvantage,  but  certain  pathogenic 
bacteria  will  not  grow  when  there  is  a  trace  of  acid  present.  The 


m 


next  step  consists  in  clarifying  the  nutrient  medium.     It  is  allowed 
tooool  to  about  50°  C.,  and  an  egg,  previously  broken  into  one 
hundred  grammes  of  water,  is  gradually  added  while  stirring  the 
liquid  with  a  -lass  rod.     A  whole  egg  is  used  for  a  litre  of  the  solu- 
Heat  is  again  applied  and  the  solution  is  kept  at  the  boiling 
point  for  about  ten  minutes,  during  which  time  the  egg  albumen  is 
precipitated  and  carries  down  with  it  all  insoluble  particles,  which 
without  this  clarifying  process  would  have  interfered  with  the  trans- 
parency of  the  medium,  even  when  carefully    filtered.     The    hot 
oiution   is  thru  filh'red.    A  hot-water  funnel  (Fig.  17)  is  usually 
•mploy«Ml.   as  tlu»  -olatin  solution  does  not   pass  through  filtering 
paper  very  rapidly,  and  when  cooled  to  near  the  point  of  solidifying 
ceases  to  pass. 


CULTURE   MEDIA.  43 

The  advantages  of  the  gelatin  medium  are  that  it  is  perfectly 
transparent,  that  it  is  easily  melted  for  making  "plates/'  and  that 
many  bacteria  exhibit  in  it  special  characters  of  growth  by  which  they 
may  be  differentiated  from  others  which  resemble  them  in  form. 
The  principal  disadvantage  is  the  low  melting  point,  which  prevents 
us  from  making  use  of  this  medium  for  cultivating  bacteria  in  an  in- 
cubating oven  at  a  higher  temperature  than  about  22°  C.  for  ten-per- 
cent gelatin. 

This  disadvantage  is  overcome  by  using  agar-agar  instead  of 
gelatin.  This  is  prepared  in  Japan  and  other  Eastern  countries 
from  certain  species  of  gelatinous  algae.  It  comes  to  us  in  the  form 
of  bundles  of  dried  strips,  which  form  a  stiff  jelly  when  dissolved  in 
water  in  the  proportion  of  one  to  two  per  cent.  This  jelly  remains 
solid  at  a  temperature  of  40°  C.  and  above.  It  was  first  employed 
by  Hesse,  one  of  Koch's  collaborators  in  the  office  of  the  imperial 
board  of  health  of  Berlin.  Koch,  who  was  in  search  of  a  trans- 
parent jelly  which  would  stand  the  temperature  required  for  the  cul- 
tivation of  certain  pathogenic  bacteria  (37°  to  38°  C.),  quickly  recog- 
nized its  value  and  introduced  it  into  general  use. 

The  agar-agar  jelly  is  more  difficult  to  filter  than  the  gelatin 
medium,  and  some  skill  is  required  in  order  to  obtain  a  transparent 
solution.  It  will  bear  long  boiling  without  losing  its  quality  of 
forming  a  stiff  jelly.  From  ten  to  twenty  grammes  are  added  to  a 
litre  of  flesh  infusion,  or  we  may  make  a peptonized  agar  in  accor- 
dance with  the  following  formula  which  is  given  by  Salomonson  : 
Add  to  one  litre  of  distilled  water  five  grammes  Liebig's  extract, 
thirty  grammes  peptone,  five  grammes  cane  sugar,  fifteen  grammes 
agar.  Cook  for  an  hour,  render  slightly  alkaline,  and  cool  to  below 
60°  C.  Clarify  and  cook  again  for  an  hour  or  more. 

Glycerin-agar  is  made  by  adding  five  per  cent  of  glycerin  to 
the  peptonized  agar  made  by  the  above  formula  or  by  the  use  of  the 
flesh-peptone  infusion.  This  is  a  very  favorable  medium  for  the  cul- 
tivation of  the  tubercle  bacillus — first  used  by  Roux  and  Nocard. 

Agar-gelatin,  a  medium  which  has  recently  come  into  favor  and 
is  said  to  be  very  useful,  as  it  resembles  gelatin  in  transparency  and 
has  a  considerably  higher  melting  point  than  ten-per-cent  gelatin,  is 
made  by  adding  fifty  grammes  of  gelatin  and  7. 5  grammes  of  agar 
to  a  litre  of  flesh-peptone  solution.  Care  should  be  taken  not  to  cook 
this  longer  than  is  necessary. 

In  making  all  of  these  agar  culture  media  the  main  difficulties 
encountered  result  from  the  difficulty  of  dissolving  the  agar  and  the 
slowness  with  which  the  solution  passes  through  filtering  paper. 
These  difficulties  are  best  met  as  follows  :  Break  up  the  sticks  of  agar 
into  small  fragments  and  allow  them  to  soak  in  cold  water  for  twenty- 


44 


CULTURE  MEDIA. 


four  hours.  Pour  off  the  water  and  add  the  flesh-peptone  solution. 
Boil  for  several  hours  until  the  agar  is  completely  dissolved.  Neu- 
tralize hy  adding  gradually  a  solution  of  carbonate  of  soda  (or  render 
slightly  alkaline).  Filter. 

The  last  operation  is  the  most  troublesome,  and  various  plans 
have  been  proposed  to  avoid  the  tedious  filtration  through  filtering 
paper  in  a  hot- water  filter.  A  method  which  gives  satisfactory  re- 
sults is  to  place  the  filter  containing  the  hot  agar  solution,  and  the 
flask  which  is  to  receive  the  filtrate,  in  a  steam  sterilizing  apparatus, 
where  it  is  left  in  an  atmosphere  of  streaming  steam  until  the  filtra- 


Fio.  18. 

tion  is  completed.  Or  the  solution  may  be  put  in  a  tall  jar  and  left 
in  the  steam  sterilizer  for  several  hours  until  it  is  clear  as  a  result  of 
sedimentation.  The  clear  solution  is  then  obtained  by  decantation. 
Or  by  conducting  the  operation  in  a  tall  cylindrical  vessel,  and  al- 
lowing sedimentation  to  occur  in  the  steam  sterilizer  and  the  agar 
subsequently  to  solidify  by  cooling,  the  cylinder  of  jelly  may  be  re- 
moved from  the  jar  and  the  part  containing  the  sediment  can  be  cut 
away.  The  transparent  portion  is  then  melted  again  and  distributed 
in  test  tubes  for  use. 

In  the  present  volume  we  frequently  refer  to  the  nutrient  medium 
madr  hy  adding  one  to  two  per  cent  of  apir-agar  to  the  standard 
tl<  >h-peptone  solution  as  **  nutrient  agar ''  or  simply  as  "  agar." 


CULTURE   MEDIA.  45 

The  following  method  of  filtering  agar  has  recently  (1890)  been 
proposed  by  Karlinsky.  It  is  a  modification  of  the  method  previously 
described  by  Jakobi  and  depends  upon  the  use  of  pressure. 

In  Fig.  18,  a  is  a  cylindrical  vessel  of  tin,  which  is  closed  above  by 
a  perforated  rubber  cork,  through  which  is  passed  a  glass  tube,  b. 
This  is  enclosed  in  a  larger  tin  cylinder,  c,  which  contains  water, 
which  may  be  kept  hot  by  placing  an  alcohol  lamp  under  the  pro- 
jecting arm  d.  The  central  cylinder  has  a  tube,  e,  passing  through 
the  bottom  of  the  hot-water  cylinder,  and  which  is  provided  with  a 


FIG.  10. 


stopcock  for  drawing  off  the  filtered  solution.  Before  pouring  the 
hot  agar  solution  into  the  cylinder  a,  a  cotton  filter  about  ten  centi- 
metres thick  is  placed  at  the  bottom  of  this  cylinder  and  hot  water 
is  poured  upon  it  while  the  stopcock  of  the  outlet  tube  is  open.  This 
washes  out  the  cotton  and  prepares  the  filter  for  the  agar  solution. 
The  apparatus  is  supported  upon  a  tripod,  not  shown  in  the  figure. 
Filtration  is  said  to  occur  rapidly  when  the  air  in  the  central  cylinder 
is  compressed  by  means  of  the  hand  bellows  attached  to  the  tube  b. 
Unna  (1891)  has  devised  a  filtering  apparatus  for  agar  which  is 
shown  in  Fig.  19.  In  this  the  pressure  of  steam  is  utilized.  A  hollow 


4»;  CULTURE  MEDIA. 

sphere  of  copper,  supported  upon  a  tripod,  is  so  constructed  that  an 
upper  hemispherical  segment  can  be  removed  to  give  access  to  the 
interior.  An  opening  at  the  bottom  contains  a  perforated  rubber 
cork,  through  which  the  stem  of  an  enamelled  iron  funnel  passes. 
A  simple  filter  of  filtering  paper  is  used  in  this  funnel,  and  this  is 
filled  to  a  depth  of  two  centimetres  with  well-burned  kieselgur  (dia- 
tomaceous  earth  in  which  the  organic  matter  has  been  destroyed  by 
heat).  The  hot  solution  of  agar  is  poured  into  the  funnel,  and  hot 
water  into  the  space  between  it  and  the  copper  vessel ;  this  must  not 
come  too  near  the  top  of  the  funnel — not  nearer  than  three  centi- 
metres. The  hemispherical  cover  is  then  secured  in  its  place  by 
means  of  a  clamp  screw  shown  in  the  figure.  By  placing  a  Bunsen 
burner  under  the  projecting  arm  the  water  is  made  to  boil  and  a 
sufficient  steam  pressure  secured.  A  small  stopcock  attached  to  the 
cover  of  the  copper  vessel  permits  the  escape  of  steam  if  the  pressure 
is  too  great.  According  to  Unna,  solutions  containing  as  much  as 
three  per  cent  of  agar  can  be  filtered  by  means  of  this  apparatus,  and 
a  litre  of  two-per-cent  agar  will  pass  through  it  in  about  two  hours. 

Schultz'  Rapid  Method  of  Preparing  Nutrient  Agar- Agar.— 
Place  one  thousand  five  hundred  cubic  centimetres  of  water  in  an  en- 
amelled iron  pot;  add  eighteen  grammes  of  agar-agar,  broken  in  small 
pieces,  and  place  upon  a  gas  stove ;  boil  for  half  an  hour ;  add  while 
boiling  two  grammes  of  Liebig's  extract  of  beef ;  remove  from  fire  and 
cool  to  60°  C. ;  then  add  ten  grammes  of  dry  peptone,  five  grammes 
of  sodium  chloride,  and  the  contents  of  one  egg  beaten  up  in  a 
sufficient  quantity  of  water  to  supply  that  lost  by  evaporation ;  neut- 
ralize the  mixture  by  the  addition  of  dilute  hydrochloric  acid ;  boil 
again  for  five  or  ten  minutes;  filter  through  white  filter  paper.  If 
the  filtrate  is  not  entirely  clear  add  to  it  the  albumen  of  a  second 
egg  and  boil  until  this  is  coagulated ;  then  filter  again.  Ahvays  mois- 
ten the  filter  with  water  before  filtering  solutions  containing 
gelatin  or  agar-agar.  When  the  process  is  completed  the  amount 
of  filtered  culture  medium  should  be  about  one  thousand  cubic  centi- 
metres. 

For  serial  purposes  various  substances  are  added  to  the  above- 
described  solid  and  liquid  media.  A  favorable  addition  for  the 
.urn  >wth  of  a  considerable  number  of  bacteria  is  from  one  to  three  per 
cent  of  glucose.  The  phosphorescent  bacteria  grow  best  in  a  medium 
<  ontaining  two  to  three  per  cent  of  sodium  chloride.  The  addition 
of  three  to  four  per  cent  of  potassium  nitrate  is  made  in  conducting 
experiments  designed  to  test  the  reducing  power  of  certain  bacteria, 
by  which  this  salt  is  decomposed  with  the  production  of  nitrites. 
Acids  are  also  added  in  various  proportion  to  test  the  ability  of 
bacteria  under  investigation  to  grow  in  an  acid  medium.  From 


CULTURE   MEDIA.  47 

1 :  2,000  to  1 :  500  of  hydrochloric  acid  may  be  used  for  this  purpose. 
The  addition  of  litmus  to  milk  or  other  culture  media  is  fre- 
quently resorted  to  for  the  purpose  of  ascertaining  whether  acids  or 
alkalies  are  developed  during  the  growth  of  bacteria  under  investi- 
gation. The  addition  of  aniline  colors  which  are  variously  changed 
by  the  products  of  growth  of  certain  species  has  also  been  resorted 
to  in  the  differentiation  of  species.  Various  disinfecting  agents,  such 
as  carbolic  acid,  etc. ,  have  also  been  used  for  the  same  purpose,  and 
it  has  been  shown  by  experiment  that  some  bacteria  will  grow  in  a 
medium  containing  such  agents  in  a  proportion  which  would  entirely 
restrain  the  development  of  others. 

The  soluble  silicates  which  form  a  jelly-like  mass  have  been 
proposed  as  a  culture  medium  for  certain  bacteria  which  do  not  grow 
in  the  usual  media.  Kiihne  (1890),  Winogradsky  (1891),  and  Sles- 
kin  (1891)  have  made  experiments  which  indicate  that  this  medium 
has  considerable  value. 

Winogradsky  uses  in  the  preparation  of  his  silicate  jelly  the 
following  salts  : 

Ammonium  sulphate,  .  .  .  .  0.4  gramme. 

Magnesium  sulphate,  .  .  .  .  0.05 

Potassium  phosphate,          ....  0.1 

Calcium  chloride,  ....  a  trace. 

Sodium  carbonate,  .  .  .  0.6  to  0.9  gramme. 

Distilled  water,  .  .  .  .  100  grammes. 

To  this  he  adds  a  solution  of  silicic  acid.  According  to  Kiihne,  a 
solution  containing  3.4  per  cent  of  silicic  acid  and  having  a  specific 
gravity  of  1.02  may  be  preserved  in  a  liquid  condition.  To  this  the 
salts  are  added  in  greater  or  less  amount,  according  to  the  consis- 
tence desired. 

Sleskin  states  that  a  suitable  jelly  is  formed  by  the  addition  of 
1.15  to  1.45  per  cent  of  the  salts,  and  recommends  that  concentrated, 
sterilized  solutions  be  added  to  the  acid.  He  dissolves  separately,  in 
as  little  water  as  possible,  the  sulphates,  the  potassium  phosphate 
and  sodium  carbonate,  and  the  calcium  chloride. 

The  use  of  a  culture  medium  containing  an  extract  from  the  je- 
quirity  seeds  has  been  recommended  by  Kaufmann  (1891),  who  has 
found,  by  experimenting  upon  various  bacteria,  that  such  a  medium 
is  useful  in  differentiating  species. 

Thejequirity  solution,  which  may  be  used  as  a  liquid  medium 
or  may  be  employed  in  the  preparation  of  nutrient  gelatin  or  agar,  is 
prepared  as  follows  :  Ten  grammes  of  jequirity  seeds  are  bruised  in 
a  mortar  and  the  shells  removed  ;  they  are  then  placed  in  one  hun- 
dred cubic  centimetres  of  water  and  cooked  for  two  hours  in  the  steam 
sterilizer  ;  after  allowing  the  infusion  to  cool  it  is  filtered.  The  fil- 
tered liquid  has  a  pale-yellow  color  and  a  neutral  or  slightly  alkaline 


48 


CULTURE   MEDIA. 


reaction.  Certain  bacteria  grow  in  this  solution  without  producing 
any  change  in  its  color  ;  others,  which  produce  an  acid  reaction, 
cause  it  to  be  decolorized  ;  others,  which  produce  an  alkaline  reac- 
tion of  the  medium,  change  the  color  to  green. 

Cooked  Potato.  —  Schroter  first  used  cooked  potato  as  a  culture 
medium  for  certain  chromogenic  bacteria  (1872),  and  Koch  subse- 
quently called  attention  to  the  great  value  of  potato  cultures  for 
differentiating  species.  His  plan  of  preparing  potatoes  is  as  follows  : 
Sound  potatoes  are  chosen  in  which  the  epidermis  is  intact.  These 
are  thoroughly  washed  and  scrubbed  with  a  brush  to  remove  all 
dirt.  The  "  eyes"  and  any  bruised  or  discolored  spots  are  removed 
with  a  sharp-pointed  knife.  They  are  again  thoroughly  washed  in 
water,  and  are  then  placed  for  an  hour  in  a  bath  containing 
mercuric  chloride  in  the  proportion  of  1  :  500,  to  thoroughly  disinfect 
the  surface.  They  are  then  placed  in  a  steam  sterilizer  for  about 
three-quarters  of  an  hour,  and  after  an  interval  of  twenty-four  hours 


A 


Fro.  80. 


Flo.  21. 


Fio.  22. 


are  again  steamed  for  fifteen  minutes.  It  is  well  to  wrap  each 
potato  in  tissue  paper  before  placing  it  in  the  bichloride  bath,  and  to 
leave  it  in  this  protecting  envelope  until  it  is  placed  in  the  glass  dish 


CULTURE  MEDIA.  49 

in  which  it  is  preserved  from  contamination  by  atmospheric  germs 
after  being  inoculated  with  some  particular  microorganism.  Just 
before  such  inoculation  the  potato  is  cut  in  halves  with  a  sterilized 
(by  heat)  table  knife.  The  bacteria  to  be  cultivated  are  placed  upon 
the  cut  surface  and  the  potato  is  preserved  in  a  glass  dish  (Fig.  20). 

A  more  convenient  method,  and  one  which  secures  the  potato  more 
effectually  from  atmospheric  organisms,  is  to  cut  a  cylinder,  about 
an  inch  in  diameter,  from  a  sound  potato,  by  means  of  a  tin  instru- 
ment resembling  a  cork  borer  or  apple  corer.  This  cylinder  is  cut 
obliquely  into  two  pieces  having  the  form  shown  in  Fig.  22,  and 
each  piece  is  placed  in  a  large  test  tube  having  a  cotton  air  filter,  in 
which  it  is  sterilized.  This  method,  first  employed  by  Bolton,  has 
been  slightly  modified  by  Roux,  who  recommends  that  a  receptacle 
for  catching  the  water  which  separates  during  the  sterilizing  process 
be  formed  by  making  a  constriction  around  the  test  tube  an  inch 
above  its  lower  extremity.  This  is  done  by  the  use  of  a  blowpipe. 
The  cylinder  of  potato  rests  upon  the  constricted  portion  of  the  tube, 
as  shown  in  Fig.  21. 

Sometimes  a  potato  paste  is  employed.  The  potatoes  are  boiled 
for  an  hour  and  the  skins  removed,  after  which  they  are  mashed 
with  a  little  sterilized  water,  placed  in  suitable  plates,  and  sterilized 
by  exposure  for  half  an  hour  on  three  successive  days  in  the  steam 
sterilizer.  Bread  paste  may  be  made  in  the  same  way,  and  is  a  very 
favorable  medium  for  the  growth  of  certain  bacteria  and  also  for  the 
common  moulds. 


VI. 
STERILIZATION  OF  CULTURE  MEDIA. 

A  MOST  important  part  of  bacteriological  technology  consists  in 
the  sterilization  of  the  various  culture  media  employed.  A  sterile 
medium  is  essential  for  maintaining  a  pure  culture,  and  we  can  only 
obtain  an  exact  knowledge  of  the  biological  characters  of  a  species 
by  studying  its  growth  in  various  media,  its  physiological  reactions, 
its  pathogenic  power,  etc.,  independently  of  all  other  microorgan- 
isms— i.e.,  in  pure  cultures. 

We  may  sterilize  a  culture  medium  either  by  heat  or  by  filtration 
through  a  substance  which  does  not  permit  bacteria  to  pass.  The 
last-mentioned  method  is  useful  for  certain  special  purposes  ;  but,  in 
general,  sterilization  of  culture  media,  and  of  the  vessels  in  which 
they  are  preserved,  is  effected  by  heat. 

The  scientific  use  of  heat  as  an  agent  for  sterilizing  our  culture 
media  depends  upon  a  knowledge  of  the  thermal  death-point  of  the 
various  microorganisms  which  are  liable  to  be  present  in  them,  and 
upon  various  facts  relating  to  the  manner  in  which  heat  is  applied. 
All  this  has  been  determined  by  experiment,  and  before  giving 
practical  directions  for  sterilization  it  will  be  well  to  consider  the 
experimental  data  upon  which  our  methods  are  based. 

As  a  rule,  bacteria  which  do  not  form  spores  are  killed  at  a  com- 
paratively low  temperature.  Thus,  in  a  series  of  experiments  made 
by  the  writer  upon  the  thermal  death-point  of  various  pathogenic 
organisms,  the  pus  cocci  were  found  to  be  the  most  resistant,  and  all 
of  these  were  killed  by  exposure  for  ten  minutes  to  a  temperature 
of  62°  C.  (143.6°  F.).  There  are  several  species  of  bacteria  known, 
however,  which  not  only  are  not  killed  by  this  temperature,  but  are 
able  to  grow  and  multiply  at  a  temperature  of  65°  to  70°  C.  (Miquel, 
Van  Tieghem,  Globig).  But  it  is  safe  to  say  that  exposure  to  a 
boiling  temperature  for  a  minute  or  two  will  infallibly  destroy  all 
microorganisms  in  the  absence  of  spores,  when  they  are  in  a  moist 
condition  or  moist  heat  is  used — i.e.,  when  they  are  directly  ex- 
posed to  the  action  of  boiling  water  or  of  steam.  The  power  of  dry 
heat  to  destroy  microorganisms  in  a  desiccated  condition  is  a  differ- 
ent matter  and  will  require  special  consideration. 


STERILIZATION   OF   CULTURE   MEDIA.  51 

The  spores  of  bacilli  have  a  much  greater  resisting  power,  and 
the  vitality  of  some  of  these  reproductive  bodies,  from  known  spe- 
cies, is  not  destroyed  by  a  boiling  temperature  maintained  for  sev- 
eral hours.  Thus  Globig  found  that  the  spores  of  a  certain  bacillus 
from  the  soil — his  "  red  potato  bacillus  " — required  six  hours'  exposure 
to  streaming  steam  in  order  to  destroy  it.  Steam  under  pressure,  at 
a  temperature  of  115°  C.,  killed  it  in  half  an  hour  ;  at  125°  C.  in  five 
minutes.  This  extreme  resisting  power  is  exceptional,  however, 
and  many  spores  are  destroyed  in  a  few  minutes  by  the  boiling  tem- 
perature of  water. 

In  practice  we  assume  that  some  of  the  more  resistant  spores, 
which  are  frequently  present  in  the  atmosphere,  may  have  fallen 
into  our  culture  material,  and  to  insure  its  sterilization  we  subject  it 
to  a  temperature  which  can  be  depended  upon  to  destroy  these  ;  or 
we  resort  to  the  method  of  discontinuous  heating.  This  method 
was  first  employed  by  Tyndall  (1877),  and  is  now  in  general  use  in 
the  bacteriological  laboratories  of  Germany,  having  been  adopted  by 
Koch  and  his  pupils ;  while  in  France  a  single  sterilization  by  means 
of  steam  under  pressure,  securing  a  higher  temperature,  is  still  the 
favorite  method  with  many. 

In  the  method  by  discontinuous  heating  we  subject  the  culture 
material  for  a  short  time  to  the  temperature  of  boiling  water,  thus 
destroying  all  bacteria  in  the  vegetative  stage.  After  an  interval, 
usually  of  twenty-four  hours,  we  repeat  the  operation  for  the  pur- 
pose of  destroying  those  which  in  the  meantime  have  developed 
from  spores  which  may  have  been  present.  Again  the  material  is 
put  aside,  and  after  twenty-four  hours  it  is  again  heated  to  the 
boiling  point.  This  is  usually  repeated  from  three  to  five  times. 
The  object  in  view  is  to  kill  the  growing  bacteria  which  are  de- 
veloped from  spores  which  were  present ;  and,  as  a  matter  of  expe- 
rience, we  find  that  this  method  of  sterilization  is  more  reliable  than 
a  single  prolonged  boiling,  unless  this  be  effected  at  a  higher  tem- 
perature than  that  of  boiling  water  at  the  ordinary  pressure  of  the 
atmosphere.  Discontinuous  heating  is  especially  useful  for  the  sterili- 
zation of  liquids  which  would  be  injured  by  prolonged  boiling — as  is 
the  case  with  solutions  of  gelatin — or  which  are  coagulated  by  the 
boiling  temperature.  By  means  of  a  water  bath,  the  temperature 
of  which  is  regulated  automatically,  we  may  conduct  the  operation 
at  any  desired  degree.  Thus  in  sterilizing  blood  serum  we  use  a 
temperature  a  little  below  that  at  which  coagulation  occurs  (about 
70°  C.). 

Test  tubes,  flasks,  and  apparatus  of  various  kinds  are  commonly 
sterilized  by  dry  heat  in  a  hot-air  oven.  This  is  usually  made  of 
sheet  iron,  with  double  walls,  and  shelves  for  supporting  the  articles 


£2  STERILIZATION  OP  CULTURE  MEDIA. 

to  be  sterilized.  The  form  shown  in  Fig.  23  is  commonly  used  in 
bacteriological  laboratories. 

It  must  be  remembered  that  a  much  higher  temperature  is  re- 
quired for  the  destruction  of  microorganisms  when  dry  heat  is  em- 
ployed than  is  the  case  with  moist  heat.  The  experiments  of  Koch 
and  Wolffhugel  (1881)  show  that  a  temperature  of  120°  to  128°  C. 
(248°  to  262°  F.)  is  required  to  destroy  the  spores  of  mould  fungi,  and 
micrococci  or  bacilli  in  the  absence  of  spores.  For  the  spores  of  ba- 
cilli a  temperature  of  140°  C.  (284°  F.),  maintained  for  three  hours, 
was  required. 

In  practice  we  usually  maintain  a  temperature  of  about  150°  C. 


Fio.28. 

(302°  F.)  for  an  hour  or  more;  and  it  is  customary  to  sterilize  all 
test  tubes  and  flasks,  which  are  to  be  used  as  receptacles  for  culture 
media,  in  the  hot-air  sterilizer.  This  procedure  could  no  doubt,  how- 
ever, be  dispensed  with  in  many  cases  and  reliance  be  placed  upon 
the  sterilization  of  the  flask,  together  with  its  contents,  in  the  steam 
sterilizer,  especially  with  such  culture  media  as  are  not  injured  by 
long  exposure  to  a  boiling  temperature — e.g.,  bouillon  and  agar-agar. 
When  we  propose  to  cultivate  aerobic  bacteria,  or  such  as  require 
oxygen  for  their  development,  a  cotton  air  filter  is  placed  in  the 
mouth  of  each  test  tube  and  flask  before  it  is  sterilized  in  the  hot-air 
oven.  This  is  a  loose  plug  of  cotton,  pushed  into  the  neck  of  the 
flask  for  an  inch  or  more,  and  projecting  from  its  mouth  for  a  short 
distance.  These  cotton  filters  should  fill  the  tube  completely  and 


STERILIZATION  OF  CULTURE  MEDIA. 


53 


uniformly,  but  should  not  be  packed  so  closely  that  there  is  difficulty 
is  removing  them. 

Steam  Sterilizers. — Steam  at  the  ordinary  pressure  of  the  atmo- 
sphere has  the  same  temperature  as  boiling  water,  and  in  practice  is 
preferable  to  a  water  bath  for  several  reasons.  The  form  of  steam 
sterilizer  adopted  by  Koch,  after  extensive  experiments  made  in  col- 
laboration with  Loffler  and  Gaffky,  is  now  generally  used  in  bacte- 
riological laboratories.  This  is  shown  in  Fig.  24.  It  consists  of  a 
cylindrical  vessel  of  zinc  which  is  covered  with  a  jacket  of  felt. 
The  cover,  also  covered  with  non-conducting  material,  has  an  aper- 
ture at  the  top  for  the  escape  of  steam.  A  glass  tube,  which  is  in 
communication  with  the  interior  of  the  vessel,  serves  to  show  the 


FIG.  24. 


FIG.  25. 


height  of  the  water  when  the  apparatus  is  in  use.  The  bottom  of 
the  cylindrical  vessel  should  be  of  copper.  A  Bunsen  burner  having 
three  jets  will  commonly  be  required  to  keep  the  water  in  ebullition 
and  the  upper  part  of  the  steam  sterilizer  filled  with  "live  steam, " 
which  should  escape  freely  from  the  aperture  in  the  cover  to  insure 
a  temperature  of  100°  C.  in  the  steam  chamber.  A  perforated  zinc 
or  copper  shelf  in  the  interior  of  the  cylinder  serves  to  support  the 
flasks,  etc.,  which  are  to  be  sterilized.  Usually  they  are  lowered 
into  the  cylinder  in  a  light  wire  basket,  or  tin  pail  with  perforated 
bottom,  of  proper  diameter  to  slip  easily  into  the  sterilizer. 

Fig.  25  is  a  sectional  view  of  this  sterilizer. 

The  steam  sterilizer  shown  in  Fig.  26  '  is  an  American  invention, 

1  The  Arnold  steam  sterilizer,  manufactured  at  Rochester,  N.  Y. 


54  STERILIZATION  OF  CULTURE  MEDIA. 

which  answers  the  purpose  admirably,  and  which  has  the  advantage 
of  getting  up  steam  very  quickly  and  also  of  using  comparatively 

little  gas. 

The  use  of  steam  under  pressure,  by  which  higher  temperatures 
are  obtained,  requires  a  more  expensive  apparatus,  made  on  the 
principle  of  Papin's  digester.  The  form  manufactured  by  Miincke 
is  one  of  the  best.  This  is  shown  in  Fig.  27.  It  is  provided  with  a 
pressure  gauge  and  a  safety  valve.  A  single  sterilization  in  this  ap- 
paratus, at  a  temperature  of  115°  C.,  for  half  an  hour,  will  usually 


Fio.  26. 


FIQ.  27. 


suffice,  and  for  liquid  culture  media  or  for  agar-agar  this  method  is 
entirely  satisfactory  ;  but  a  gelatin  medium  which  is  exposed  to  this 
temperature  loses  its  property  of  forming  a  jelly  at  20°  to  22°  C.,  and 
consequently  its  value  as  a  solid  culture  medium.  In  practice  the 
simpler  form  of  apparatus  in  which  streaming  steam  is  used  will  be 
found  to  answer  every  requirement.  To  insure  sterilization  with 
this  it  is  customary  to  resort  to  discontinuous  heating,  as  heretofore 
described.  The  standard  flesh-peptone-gelatin  medium  should,  as 
a  rule,  be  subjected  to  a  temi>erature  of  100°  C.  for  ten  minutes,  at 
intervals  of  twenty-four  hours,  four  days  in  succession.  Bouillon, 
H«-*h  int'iiMMiis.  and  a^ar-agiir  jolly  may  be  steamed  for  an  hour  at  a 
time  two  or  three  days  in  succession. 


STERILIZATION   OF  CULTURE   MEDIA.  55 

It  is  always  advisable  to  test  the  sterilization  of  culture  material 
before  making  use  of  it.  This  is  done  by  placing  it  for  a  few  days 
in  an  incubating  oven  at  30°  to  35°  C.  If  a  considerable  quantity  of 
material  in  test  tubes  has  been  prepared  at  one  time,  it  will  be  suffi- 
cient to  put  a  few  tubes  in  the  incubating  oven  to  test  sterilization. 

Failure  to  make  this  test  often  leads  to  serious  complications  in 
experimental  investigations.  A  laboratory  sometimes  becomes  in- 
fected with  resistant  spores,  which  are  not  all  destroyed  by  the  usual 
methods  of  sterilization,  and  these  may  not  develop  until  some  time 
has  elapsed  after  the  supposed  sterilization. 

Sterilization  of  Blood  Serum. — Blood  serum  which  has  been 
collected  in  test  tubes  or  small  flasks,  as  heretofore  directed,  is 


FIG.  28. 

sterilized  in  a  water  bath  at  60°  C.  (140°  F.)  by  the  method  of  dis- 
continuous heating.  It  is  usually  left  in  the  hot-water  bath  for 
about  an  hour,  and  this  is  repeated,  at  intervals  of  twenty-four  hours, 
for  five  to  seven  days.  This  rather  tedious  process  may  be  avoided 
by  collecting  the  serum  in  the  first  instance  with  proper  precautions 
to  prevent  it  from  becoming  contaminated  with  atmospheric  organ- 
isms. A  special  apparatus  was  devised  by  Koch  for  sterilizing  blood 
serum,  but  an  improvised  hot-water  bath  which  is  regulated  to  a 
temperature  of  60°  C.  by  an  automatic  thermo-regulator  will  answer 
the  purpose.  After  being  sterilized  the  serum  is  solidified  by  careful 
exposure  to  a  temperature  of  about  68°  C.,  which  causes  it  to  co- 
agulate, forming  a  transparent,  jelly-like  mass.  When  coagulated 
at  a  higher  temperature  it  becomes  opaque.  The  time  required  for 
this  operation  varies  from  half  an  hour  to  an  hour,  and  it  is  best  to 
remove  the  tubes  from  the  receptacle  in  which  they  are  exposed  to 


56 


STERILIZATION   OF  CULTURE   MEDIA. 


heat  as  soon  as  the  serum  is  solidified.  Koch's  apparatus  for  coagu- 
lating blood  serum  is  shown  in  Fig.  28.  It  is  customary  to  place  the 
test  tubes  in  an  oblique  position,  so  that  a  large  surface  may  be  ex- 
posed upon  which  to  cultivate  the  tubercle  bacillus  or  whatever 
microorganism  may  be  under  investigation.  A  form  of  apparatus 
designed  for  both  sterilizing  and  coagulating  blood  serum  is  shown 
in  Fig.  29.  It  is  manufactured  by  Miincke  in  accordance  with  the 
directions  of  Hueppe,  and  special  precautions  have  been  taken  to  se- 
cure a  uniform  temperature  in  all  parts  of  the  air  chamber.  We 


FIG.  29. 

may  remark  that  since  it  has  been  shown  by  Roux  and  Nocard  that 
the  tubercle  bacillus  grows  very  well  in  agar-agar  jelly  to  which 
five  per  cent  of  glycerin  has  been  added,  blood  serum  is  not  so 
largely  used  as  a  culture  medium  in  bacteriological  laboratories. 

Sterilization  by  Filtration.— This  method  is  especially  useful 

for  separating  the  soluble  substances  contained  in  a  liquid  culture  of 

bacteria  from  the  living  cells.     It  has  been  demonstrated  that  several 

e  most  important  pathogenic  bacteria  produce  toxic  substances 

during  their  growth  which  may  cause  the  death  of  susceptible  ani- 

mdependently  of  the  living  bacteria;  and  this  demonstration 


STERILIZATION   OF   CULTURE   MEDIA.  57 

has  been  made  either  by  sterilizing  a  pure  culture  by  means  of  heat, 
or  by  separating  the  bacteria  from  the  culture  liquid  by  filtration. 
Some  of  these  toxic  products  of  bacterial  growth  are  destroyed  by  a 
comparatively  low  temperature ;  the  method  of  sterilization  by  fil- 
tration is  therefore  very  important  in  researches  relating  to  the 
composition  and  pathogenic  power  of  these  soluble  products.  Pas- 
teur, in  his  earlier  experiments,  used  plaster  of  Paris  as  a  filter,  and 


Fig.  30. 


subsequently  resorted  to  the  use  of  unglazed  porcelain,  through 
which  a  liquid  may  be  forced  by  pressure,  but  which  does  not  per- 
mit of  the  passage  of  suspended  particles,  however  small. 

As  the  porcelain  filter  is  the  most  reliable  and  convenient  for 
accomplishing  the  object  in  view,  we  shall  not  describe  other  methods 
of  filtration  which  have  been  proposed  and  successfully  used.  The 
porcelain  used  is  a  very  fine  paste,  manufactured  at  Sevres,  which  is 
moulded  into  cylinders  (bougies)  of  the  form  proposed  by  Chamber- 
land  and  baked  at  a  high  temperature. 


58 


STERILIZATION  OF  CULTURE  MEDIA. 


In  Fig.  30  the  Pasteur-Chamberland  filter  is  shown  as  arranged 
for  the  filtration  of  water.  A  is  the  hollow  porcelain  cylinder,  which 
is  enclosed  in  a  metal  case,  D.  The  metal  case  is  tightly  clamped 
against  a  projecting  shoulder  at  the  lower  part  of  the  porcelain  filter, 
a  ring  of  rubber  being  interposed  to  secure  a  tight  joint.  When 
water  under  pressure  is  admitted  to  the  space  E,  between  the  cylin- 
der of  porcelain  and  the  metal  case,  it  slowly  filters  through,  and, 
running  down  the  inner  wall  of  the  filter,  escapes  at  B  into  a  recep- 
tacle placed  to  receive  it.  If  we  fill  the  space  E  with  a  liquid  cul- 
ture of  bacteria  and  apply  sufficient  pressure  (one  or  two  atmo- 
spheres), a  clear  filtrate  is  obtained  which  is  entirely  sterile  if  the 
porcelain  filter  is  sound  and  made  of  proper  material.  After  the 


Fio.  31. 

filter  has  been  in  use  for  some  time,  however,  it  may  permit  the  pas- 
sage of  bacteria,  and  it  will  be  necessary  to  subject  it  to  a  high  tem- 
perature for  the  purpose  of  destroying  all  organic  matter  contained 
in  the  porous  porcelain. 

We  may  use  the  Chamberland  filter  without  a  metal  case  by  im- 
mersing it  in  a  cylindrical  glass  vessel  containing  the  liquid  to  be  fil- 
tered, as  shown  in  Fig.  31.  The  porcelain  cylinder  is  connected  with 
an  aspirator  bottle,  a,  and  a  small  Erlenmeyer  flask,  6,  is  interposed 
to  catch  tlic  lilt  rate  \vhen  it  overflows  from  the  interior  of  the  filter. 
Of  course  all  the  necessary  precautions  must  be  taken  with  refeivmv 
to  the  sterilization  of  the  interior  of  the  bougie,  of  the  flask  b,  and  of 
the  rubber  tube  connecting  the  two. 

Another  arrangement  of  the  Pasteur-Chamberland  filter  for  labora- 
tory purposes  is  shown  in  Fig.  32.  In  this  form  of  apparatus  a 


STERILIZATION   OF   CULTURE   MEDIA. 


59 


receptacle,  R,  is  provided  for  the  liquid  to  be  filtered,  and  a  pump  for 
compressing  air  is  attached  to  it  by  a  rubber  tube.  Instead  of  this 
pump,  water  pressure  may  be  used  indirectly  by  attaching  a  strong 
bottle  to  the  water  supply  and  allowing  it  to  fill  slowly  with  water, 
and  at  the  same  time  to  force  out  the  air  through  a  tube  connected 
with  the  filtering  apparatus.  For  this  purpose  the  bottle,  having  a 
capacity  of  a  quart  or  more,  should  be  provided  with  a  rubber  stop- 
per through  which  two  short  tubes  are  passed.  One  of  these  is  con- 
nected with  the  water  supply  and  the  other  with  the  filter.  Of 
course  this  is  only  practicable  when  a  water  supply  with  sufficient 
pressure  is  available. 


FIG.  32. 

As  a  rule,  filtration  cannot  be  substituted  with  advantage  for  ster- 
ilization by  heat  in  the  preparation  of  culture  media.  Albuminous 
liquids  pass  through  the  filter  with  difficulty,  and  the  process  of 
sterilization  by  discontinued  heating  will  usually  prove  more  satis- 
factory than  filtration,  which  requires  extreme  precautions  to  pre- 
vent accidental  contamination  of  the  filtered  liquid.  Moreover,  the 
filter  may  change  the  composition  of  the  medium  passed  through  it 
by  preventing  the  passage  of  colloid  and  albuminous  material  in  so- 
lution. Thus,  in  an  attempt  to  separate  blood  corpuscles  from  the 
serum  by  filtration  through  a  Chamberland  filter,  the  writer  obtained 
a  transparent  liquid  which  did  not  coagulate  by  heat — i.  e. ,  the  albu- 
minous constituents  of  the  serum  did  not  pass  through  the  filter. 


VII. 
CULTURES  IN  LIQUID  MEDIA. 

PRIOR  to  the  introduction  of  gelatinous  media  by  Koch  in  1881  f 
cultures  were  made  in  various  organic  liquids,  and  these  are  still 
largely  used,  being  for  certain  purposes  preferable  to  solid  media. 
The  method  of  preparing  and  sterilizing  the  flesh  infusions  and 
other  organic  liquids  commonly  used  has  already  been  given.  We 
are  here  concerned  with  the  various  modes  of  using  these  nutritive 
liquids  in  cultivating  bacteria. 

Flasks  and  tubes  of  various  forms  have  been  employed  by  differ- 
ent investigators,  but  the  most  useful  receptacle  for  liquid  as  well  as 
for  solid  culture  media  is  the  ordinary  test  tube.  These  are  care- 
fully cleaned,  plugged  with  a  cotton  air  filter,  sterilized  in  the  hot-air 
oven  at  150°  C.,  and  are  then  ready  to  receive  the  filtered  liquid. 
Usually  the  tube  should  not  be  filled  to  more  than  one-third  to  one- 
half  of  its  capacity.  Sterilization  of  the  culture  liquid  is  then  effected 
by  placing  the  tubes  in  the  steam  sterilizer  for  half  an  hour  on  three 
successive  days.  Before  using,  the  tubes  should  be  placed  for  a  few 
days  in  an  incubating  oven  at  30°  to  35°  C.  to  test  the  sterilization. 
This  is  especially  important  with  liquid  media,  for  if  a  single  living 
spore  is  present  it  may  give  rise  to  an  abundant  progeny,  which  will 
be  distributed  through  the  liquid  in  association  with  the  species 
which  has  been  planted.  In  solid  cultures,  on  the  contrary,  such  a 
spore  would  give  rise  to  a  colony,  which  by  its  locality  and  characters 
of  growth  would  probably  be  recognized  as  different  from  the  species 
planted,  and  consequently  accidental.  This  is  the  great  danger  in 
the  use  of  liquid  media  ;  imperfect  sterilization,  or  accidental  contami- 
nation by  atmospheric  germs,  may  lead  the  inexperienced  student 
into  serious  errors  resulting  from  the  assumption  that  the  micro- 
organisms present  in  his  cultures  are  all  derived  from  the  seed  he 
planted. 

On  1h«»  other  hand,  liquid  media  are  more  convenient  than  solid 
when  it  is  t  lie  intention  to  isolate  by  filtration  the  soluble  products  of 
hartrrial  growth;  for  injection  into  animals  to  test  pathogenic  power; 
for  experiments  on  the  germicidal  or  antiseptic  power  of  chemical 
agents,  etc. 


CULTURES   IN   LIQUID   MEDIA. 


61 


For  larger  quantities  of  liquid  than  can  be  held  in  an  ordinary 
test  tube  the  small  flasks  with  a  flat  bottom,  known  as  Erlenmeyer 
flasks,  are  very  convenient  (Fig.  33). 

In  his  earlier  researches  Pasteur  used  flasks  and  tubes  of  various 
forms,  which  served  a  useful  purpose,  but  have  been  displaced  in  his 
laboratory  by  the  simpler  form  of  apparatus  shown  in  Fig.  34. 
This  is  a  little  flask  having  a  cover  which  is  ground  to  fit  the  neck. 
This  cover  is  drawn  out  above  into  a  narrow  tube  which  admits 
oxygen  to  the  flask  through  a  cotton  air  filter.  To  obtain  access 
to  the  interior  of  the  flask  for  the  purpose  of  introducing  bacteria 
to  start  a  culture,  or  to  obtain  material  for  microscopical  examina- 
tion, the  cover  is  detached  at  the  ground  joint  by  a  gentle  twisting 
motion. 

There  is  much  less  danger  that  a  sterile  culture  liquid  will  become 


FIG.  33. 


FIG.  34. 


contaminated  during  the  momentary  removal  of  the  cover  from 
one  of  these  little  flasks,  or  of  the  cotton  plug  from  a  test  tube,  than 
is  usually  supposed.  Abundant  laboratory  experience  demonstrates 
that  such  contamination  by  bacteria  floating  in  the  atmosphere  rarely 
occurs.  The  spores  of  mould  fungi  are  commonly  more  abundant 
in  the  air,  but  even  these  do  not  very  frequently  fall  into  the  culture 
liquid  when  the  tube  is  opened  to  inoculate  it  with  the  bacteria  it  is 
proposed  to  cultivate.  This  inoculation  is  best  made  with  a  platinum 
wire,  bent  into  a  loop  at  the  free  extremity,  and  sealed  fast  into  the 
end  of  a  glass  rod  (Fig.  35).  This  is  sterilized  in  the  flame  of  a 
Bunsen  burner  or  alcohol  lamp  by  bringing  the  platinum  wire  to  a 
red  heat  and  passing  the  end  of  the  glass  rod  which  carries  it 
through  the  flame  several  times.  With  this  instrument  we  may 
transfer  a  little  drop  from  a  culture  to  the  sterile  fluid  in  another 


frt  CULTURES   IX   LIQUID   MEDIA. 

tube  for  the  purpose  of  starting  a  new  culture.  Or  we  may  start  a 
pure  culture  from  a  drop  of  blood  taken  from  the  veins  of  an  animal 
which  has  been  inoculated  with  anthrax,  or  any  similar  infectious 
disease  in  which  the  blood  is  invaded  by  a  bacterial  parasite. 

But  if  we  have  not  a  pure  culture  to  start  with  our  liquid  media 
do  not  afford  us  the  means  of  obtaining  one ;  and  if  two  or  more 
bacteria  which  resemble  each  other  in  their  morphology  are  associated 
in  such  a  culture  we  cannot  differentiate  them,  and  are  likely  to  infer 
that  we  have  a  pure  culture  of  a  single  microorganism  when  this  is 
not  really  the  case. 

But  if  we  have  pure  stock  to  start  with  we  may  maintain  pure 
cultures  in  liquid  media  without  any  special  difficulty. 

Various  characters  of  growth,  etc.,  are  to  be  observed  in  culti- 
vating different  microorganisms  in  liquid  media.  Thus  some  grow 
at  the  surface  in  the  form  of  a  thin  film  or  membranous  layer — "  my- 
coderma  " — while  others  are  distributed  uniformly  through  the  liquid, 
rendering  it  opalescent  or  more  or  less  milky  and  opaque  ;  others, 
again,  form  little  flocculi  which  are  suspended  in  the  transparent 


Fro.  35. 


fluid.  Usually,  when  active  growth  has  ceased,  the  bacteria  fall  to 
the  bottom  of  the  tube  as  a  more  or  less  abundant,  white  or  colored, 
pulverulent  or  glutinous  deposit.  In  some  cases  the  liquid  is  colored 
with  a  soluble  pigment  formed  during  the  growth  of  the  bacteria, 
and  usually  this  is  formed  most  abundantly  at  the  surface,  where 
there  is  free  access  of  oxygen.  The  reaction  of  the  medium  is  often 
changed  as  a  result  of  the  growth  of  bacteria  in  it.  From  being  neu- 
tral it  may  become  decidedly  alkaline  or  acid  in  its  reaction.  These 
changes  may  be  observed  by  adding  a  litmus  solution  before  sterili- 
zation of  the  culture  medium,  and  observing  the  change  of  color 
when  an  acid-producing  bacterium  is  under  cultivation.  The  re- 
ducing power  of  bacteria  upon  various  aniline  colors  may  also  be 
MU  died  ;  also  their  power  to  break  up  various  organic  substances 
shown  by  the  evolution  of  gas  or  other  volatile  products  which 
may  be  collected,  or  by  substances  which  remain  in  solution  and 
can  !><>  studied  by  ordinary  chemical  methods. 

Drop  Cultures. — When  we  desire  to  study  the  life  history  of  a 
microorganism  and  to  witness  its  development  from  spores,  for  ex- 
ample, its  motions,  etc.,  the  method  of  cultivation  in  a  hanging  drop 


CULTURES   IN   LIQUID   MEDIA.  63 

of  culture  fluid,  attached  to  a  thin  glass  cover  and  suspended  over  a 
circular  excavation  ground  out  of  a  glass  shoe,  is  very  useful. 
Such  a  drop  culture  may  be  left  under  the  microscope  and  kept 
under  observation  for  hours  or  days. 

pieveat  the  inoculation  of  the  drop  of  culture  liquid  with  any  other 
bacteria  than  those  which  are  to  be  studied. 

The  smiJMdk  form  of  moist  chamber  for  drop  cultures  consists  of 
an  ordinary  glass  shoe  having  a  concave  depression,  about  fifteen 
in  diameter,  ground  out  in  its  centre.  This  and  the  thin 
r.  having  been  sterflhed  by  exposure  in  the  hot-air  oven  at 
ISO0  CL  for  an  hoar  or  more,  or  by  passing  them  through  the  flame 
of  an  alcohol  lamp,  are  ready  for  use.  The  cover  glass  is  held  in 
sterile  forceps,  and  a  little  drop  of  the  culture  fluid  containing  the 
bacterium  to  be  studied  is  transferred  to  its  centre  by  means  of  the 
pfatjnmn  loop  heretofore  described.  It  is  best  to  spread  the  drop 
out  as  thin  as  possible,  and  it  may  be  inoculated,  from  a  pure  cul- 


36)  after  it  has  been  placed  upon 
he  hollow  place  in  the  glass 
to  prevent  the  entrance  of  air  and  attach 
EtOa  VMBBIM  HOMnd  (he  mtuspm  of  flba 


by  attaching  a  glass 
to  the  centre  of  a  glass  elide 

by 

In  Ranvier  s  moist  chamber  there  is  a  central  eminence  sur- 
by  a  groove  ground  into  the  glass  slide,  and  the  drop  of 

above.    Tins  affords  a  more  satisfactory  view  under  the  micro- 


TheA*tkor*C«lt«ri>  JfefltodL— In  a  paper  read  at 

1681,  the  writer  described  a  method  of  conducting  culture 


64  CULTURES  IN  LIQUID  MEDIA. 

berg's  bulbs/'  as  they  are  sometimes  called,  are  that  a  culture  me- 
dium may  be  preserved  in  them  indefinitely  and  that  they  are  easily 
transported  from  place  to  place;  whereas  test  tubes,  Pasteur's  flasks, 
and  similar  receptacles  must  be  kept  upright,  and  after  a  time  the 
culture  liquid  in  them  is  changed  in  its  composition  by  evaporation. 
They  are  also  liable  to  be  contaminated  by  the  entrance  of  mould 
fungi  when  kept  in  a  damp  place.  The  spores  of  these  fungi,  falling 
upon  the  surface  of  the  cotton  air  filter,  germinate,  and  the  myce- 
lium grows  down  through  the  cotton  into  the  interior  of  the  tube, 
where  a  new  crop  of  spores  is  quickly  formed.  It  is,  therefore,  a 
convenience  to  have  sterile  culture  liquids  always  ready  for  use  in 
a  receptacle  which  can  be  packed  in  a  box  and  transported  from 
place  to  place ;  but  for  every-day  use  in  the  laboratory  the  ordinary 


Fio.  37. 

test  tube,  with  its  cotton  air  filter,  is  the  most  economical  and  conve- 
nient receptacle  for  culture  liquids  as  well  as  for  solid  media.  With 
reference  to  the  method  of  making  and  using  these  little  flasks,  I 
quote  from  a  paper  published  in  the  American  Journal  of  the 
Medical  Sciences  in  1883  :J 

The  culture  flasks  employed  contain  from  one  to  four  fluidrachms. 
They  are  made  from  glass  tubing  of  three-  or  four- tenths  inch  diameter,  and 
those  which  the  writer  has  used  in  his  numerous  experiments  have  all  been 
**  home-made."  It  is  easier  to  make  new  flasks  than  to  clean  old  ones,  and 
they  are  thrown  away  after  being  once  used.  Bellows  operated  by  foot,  and 
a  flame  of  considerable  size — gas  is  preferable — will  be  required  by  one  who 
proposes  to  construct  these  little  flasks  for  himself.9  After  a  little  practice 
they  are  made  rapidly ;  but  as  a  large  number  are  required,  the  time  and 
labor  expended  in  their  preparation  are  no  slight  matter.  After  blowing  a 
bulb  at  the  extremity  of  a  long  glass  tube,  of  the  diameter  mentioned,  this 
is  provided  with  a  slender  neck,  drawn  out  in  the  flame,  and  the  end  of  this 

1  "  The  Germicide  Value  of  Certain  Therapeutic  Agents,"  op.  cit.,  vol.  clxx. 
"  A  glass-blower  ought  to  make  them  for  two  or  three  dollars  per  hundred. 


CULTURES  IN  LIQUID  MEDIA.  65 

is  hermetically  sealed.  Thus  one  little  flask  after  another  is  made  from  the 
same  piece  of  tubing-  until  this  becomes  too  short  for  further  use.  To  intro- 
duce a  culture  liquid  into  one  of  these  little  flasks,  heat  the  bulb  slightly, 
break  off  the  sealed  extremity  of  the  tube  and  plunge  it  beneath  the  surface 
of  the  liquid  (Fig.  37).  The  quantity  which  enters  will  of  course  depend 
upon  the  heat  employed  and  the  consequent  rarefaction  of  the  enclosed  air. 
Ordinarily  the  bulb  is  filled  to  about  one-third  of  its  capacity  with  the  cul- 
ture liquid,  leaving  it  two-thirds  full  of  air  for  the  use  of  the  microscopic 
plants  which  are  to  be  cultivated  in  it.  ...  Sterilization  is  effected  by  heat 
after  the  liquid  has  been  introduced  and  the  neck  of  the  flask  hermetically 
sealed  in  the  flame  of  an  alcohol  lamp. 

Sterilization  may  be  effected  by  boiling  for  an  hour  in  a  bath  of  paraffin 
or  of  concentrated  salt  solution,  by  which  a  temperature  considerably  above 
that  of  boiling  water  is  secured.  The  writer  is  in  the  habit  of  preparing  a 
considerable  number  of  these  flasks  at  one  time,  and  leaving  them,  in  a  suit- 
able vessel  filled  with  water,  for  twenty- four  hours  or  longer  on  the  kitchen 
stove.1 

To  inoculate  the  liquid  contained  in  one  of  these  little  flasks  with  mi- 
croorganisms from  any  source,  the  end  of  the  tube  is  first  heated  to  destroy 
germs  attached  to  the  exterior;  the  extremity  is  then  broken  off  with  steril- 
ized (by  heat)  forceps;  the  bulb  is  very  gently  heated,  so  as  to  force  out  a 
little  air,  and  the  open  end  is  plunged  into  the  liquid  containing  the  organ- 
ism to  be  cultivated  (or  into  a  vein,  or  one  of  the  solid  viscera  of  an  animal 
dead  from  an  infectious  germ  disease,  such  as  anthrax). 

Inoculation  from  one  tube  to  another  may  also  be  effected  by  means  of 
the  ordinary  platinum  wire  needle. 

Before  the  introduction  of  Koch's  plate  method  for  isolating  bac- 
teria in  pure  cultures,  certain  methods  had  been  proposed,  and  em- 
ployed to  some  extent,  which  at  present  have  a  historical  value  only. 

Thus  Klebs  (1873)  proposed  to  take  from  a  first  culture  in  which 
two  or  more  species  were  associated  a  minute  quantity,  by  means  of  a 
capillary  tube,  and  with  this  to  inoculate  a  second  culture.  By  re- 
peating this  procedure  several  times  he  expected  to  exclude  all  except 
the  species  which  was  present  in  the  greatest  abundance  and  which 
multiplied  most  rapidly  in  the  medium  employed. 

The  method  by  dilution,  first  employed  with  precision  by  Brefeld 
(1872)  in  obtaining  pure  cultures  of  mould  fungi,  and  subsequently 
by  Lister  for  the  isolation  of  bacteria,  consists  in  so  diluting  a  minute 
quantity  of  the  mixed  culture  that  the  number  of  bacteria  in  the  dilu- 
tion may  be  less  than  one  for  each  drop  of  the  liquid.  If  now  a 
single  drop  be  added  to  each  of  a  series  of  tubes  containing  a  small 
quantity  of  sterile  bouillon,  some  of  the  inoculations  made  may  give 
a  pure  culture,  as  the  drop  may  have  contained  but  a  single  vege- 
tative cell. 

Another  method  of  obtaining  a  pure  culture  in  liquid  media,  when 
several  microorganisms  are  associated  which  have  a  different  ther- 

1  Where  a  steam  sterilizer  is  at  hand  they  will  be  most  conveniently  sterilized  in 
the  usual  way,  by  subjecting  them  to  the  boiling  temperature  for  an  hour  at  a  time 
on  three  successive  days. 
5 


66 


CULTURES   IN   LIQUID   MEDIA. 


mal  death-point,  consists  in  the  application  of  heat  and  thus  destroy- 
ing all  except  the  most  resistant  species.  This  method  is  especially 
applicable  when  one  of  the  species,  only,  forms  spores.  By  subject- 
ing the  mixed  culture  to  a  temperature  which  is  sufficient  to  destroy 
all  the  vegetative  cells  in  it,  the  more  resistant  spores  are  left  and, 
under  favorable  conditions,  may  subsequently 
vegetate  and  give  us  a  pure  culture  of  the 
species  to  which  they  belong. 

Fermentation. — The  development  of  certain 
bacteria  is  attended  with  an  evolution  of  gas, 
especially  in  media  containing  grape  sugar  or 
glycerin.  For  the  determination  of  the  quantity 
and  kind  of  gas  produced  by  a  given  micro- 
organism the  fermentation  tube  recommended 
by  Theobald  Smith  has  special  advantages. 
This  is  a  bent  tube  (Eihorn's)  supported  upon 
a  glass  base  as  shown  in  the  accompanying 
figure  taken  from  the  catalogue  of  Eimer  & 
Amend.  The  graduation  shown  upon  the  up- 
right arm  is  not  essential  for  ordinary  labora- 
tory work.  A  liquid  culture  medium  containing 
one  to  two  per  cent  of  grape  sugar  is  usually 
used.  This  is  introduced  into  the  upright  arm 
of  the  fermentation  tube,  where  it  is  held  by  atmospheric  pressure. 
A  cotton  plug  is  placed  in  the  opening  of  the  short  and  bulbous  arm 
of  the  tube,  which  is  intended  as  a  receptacle  for  the  culture  liquid 
when  it  is  forced  out  of  the  closed  arm  by  the  accumulation  of  gas  at 
its  upper  extremity. 


Fio.  38. 


VIII. 
CULTURES  IN  SOLID  MEDIA. 

THE  introduction  of  solid  culture  media  in  1881  by  the  famous 
German  bacteriologist,  Robert  Koch,  inaugurated  a  new  era  in  the 
progress  of  our  knowledge  relating  to  the  bacteria.  His  methods 
enable  us  to  obtain  pure  cultures  with  ease  and  certainty,  and  to 
study  the  morphological  and  biological  characters  of  each  species 
free  from  the  complications  which  led  to  so  much  error  and  confusion 
before  these  methods  were  introduced.  We  have  already  given  an 
account  of  the  method  of  preparing  and  sterilizing  the  various  solid 
culture  media,  and  are  here  concerned  with  the  manner 
in  which  they  are  used  and  the  special  advantages  which 
they  afford. 

Koch's  flesh-peptone-gelatin,  which  contains  ten  per 
cent  of  gelatin,  is  a  transparent  jelly  which  liquefies  at 
from  22°  to  24°  C.  It  is  a  favorable  culture  medium  for 
a  great  number  of  bacteria,  and  many  species  show  de- 
finite characters  of  growth  in  this  medium  which  serve  to 
differentiate  them.  One  of  the  most  prominent  of  these 
characters  depends  upon  the  fact  that  some  bacteria  liquefy 
gelatin  and  others  do  not.  This  is  made  apparent  when 
we  make  stick  cultures — also  called  "stab  cultures." 
This  is  the  usual  manner  of  inoculating  a  solid  culture 
medium,  and  is  illustrated  in  Fig.  39.  A  platinum  needle, 
consisting  of  a  piece  of  platinum  wire  inserted  into  a  glass 
rod  which  serves  as  a  handle,  is  passed  through  the  flame 
of  an  alcohol  lamp  to  sterilize  it.  When  cooled,  which 
occurs  very  quickly,  the  point  is  introduced  into  the  ma- 
terial containing  the  bacteria  to  be  planted  in  the  gelatin 
medium.  We  may  obtain  our  seed  for  a  pure  culture  FlG  ^ 
from  a  single  colony,  from  another  stick  culture,  from  the 
blood  of  an  infected  animal,  etc.  The  point  of  the  needle  is  then 
carried  into  the  sterilized  jelly,  as  shown  in  the  figure,  care  being 
taken  to  introduce  it  in  the  central  line  and  in  a  direction  parallel 


68 


CULTURES  IN  SOLID  MEDIA. 


with  the  sides  of  the  tube.  It  is  best  always  to  hold  the  tube  in- 
verted during  the  inoculation,  and  not  to  remove  the  cotton  air  filter 
until  we  are  ready  to  make  it.  The  cotton  plug  is  then  returned  to 
its  place  and  the  platinum  needle  again  brought  to  a  red  heat  to 
destroy  any  bacteria  which  remain  attached  to  it. 

Sometimes  it  is  an  advantage  to  have  the  culture  medium  with  a 


FIG.  40. 


sloping  surface,  as  shown  in  Fig.  40.    We  may  then  draw  the  nee- 
dle over  the  surface  in  a  longitudinal  direction,  and  by  this  means 
distribute  the  seed  in  a  line  along  which  development  will  take  place. 
The  characters  of  growth  in  these  stick  cultures  in  gelatin  are 


very  various.  Non-liquefying  bacteria  may  grow  only  on  the  sur- 
face, as  at  a,  Fig.  40A ;  or  both  on  the  surface  and  along  the  line 
of  puncture,  as  at  b;  or  only  at  the  bottom,  as  at  c.  In  the  first 
case  the  microorganism  is  aerobic— that  is,  it  requires  oxygen,  and 
grows  only  in  the  presence  of  this  gas.  In  the  second  case  it  is 
not  strictly  aerobic,  but  may  grow  either  in  the  presence  of  oxygen 


CULTURES  IN   SOLID  MEDIA. 


69 


or  in  its  absence — a  facultative  anaerobic.  In  the  third  case  the 
microorganism  is  an  anaerobic,  which  cannot  grow  in  the  presence 
of  oxygen,  and  consequently  does  not  grow  upon  the  surface  of  the 
culture  medium  or  along  the  upper  portion  of  the  line  of  puncture. 

Again,  we  have  differences  as  to  the  character  of  growth  upon  the 
surface  or  along  the  line  of  puncture.  The  surface  growth  may  be 
a  little  mass  piled  up  at  the  point  where  the  needle  entered  the  gela- 
tin ;  or  it  may  form  a  layer  over  the  entire  surface,  and  this  may 
be  thin  or  thick,  dry  or  moist,  viscid  or  cream-like,  and  of  various 
colors — green,  blue,  red,  or  yellow,  of  different  shades — or  more  fre- 
quently of  a  milk-white  color. 


-JC 


k 


The  growth  along  the  line  of  puncture  also  differs  greatly  with 
different  species.  We  may  have  a  number  of  scattered  spherical 
colonies  (a,  Fig.  41),  and  these  may  be  translucent  or  opaque  ;  or  we 
may  have  little  tufts,  like  moss,  projecting  from  the  line  of  puncture 
(6,  Fig.  41)  ;  or  slender,  filamentous  branches  may  grow  out  into  the 
gelatin  (c,  Fig.  41). 

The  liquefying  bacilli  also  present  different  characters  of  growth. 
Thus  liquefaction  may  take  place  all  along  the  line  of  puncture, 
forming  a  long  and  narrow  funnel  of  liquefied  gelatin  (a,  Fig.  42) ; 
or  we  may  have  a  broad  funnel,  as  at  b  ;  or  a  cup-shaped  cavity,  as 
at  c;  or  the  upper  liquefied  portion  may  be  separated  from  that 
which  is  not  liquefied  by  a  horizontal  plane  surface,  as  at  d. 


fO  CULTURES   IN   SOLID   MEDIA. 

The  characters  of  growth  in  agar-agar  jelly  are  not  so  varied, 
but  this  medium  possesses  the  advantage  of  not  liquefying  at  a  tem- 
perature of  35°  to  38°  C.,  which  is  required  for  the  development  of 
certain  pathogenic  bacteria.  Variations  in  mode  of  growth  are 
also  manifested  in  nutrient  agar  similar  to  those  referred  to  as  pro- 
duced by  non-liquefying  bacteria  in  flesh-peptone-gelatin.  These 
relate  to  the  surface  growth  and  to  growth  along  the  line  of  punc- 
ture. One  character  not  heretofore  mentioned  consists  in  the  for- 
mation of  gas  bubbles  in  stick  cultures  either  in  gelatin  or  agar. 

Colonies. — If  we  melt  the  gelatin  or  agar  in  a  test  tube,  pour 
the  liquid  medium  into  a  shallow  glass  dish  previously  sterilized, 


FIG.  42. 

and  allow  it  to  cool  while  properly  protected  by  a  glass  cover,  we 
will  have  a  broad  surface  of  sterile  nutrient  material.  If  now  we  ex- 
pose it  to  the  air  for  ten  or  fifteen  minutes,  and  again  cover  it  and 
put  it  aside  for  two  or  three  days  at  a  favorable  temperature,  we  can 
scarcely  fail  to  have  a  number  of  colonies  upon  the  surface  of  the 
culture  medium,  which  have  been  developed  from  atmospheric  germs 
which  were  deposited  upon  it  during  the  exposure.  Each  of  these 
colonies,  as  a  rule,  is  developed  from  a  single  bacterium  or  spore, 
and  consequently  the  little  mass,  visible  to  the  naked  eye,  which  we 
call  a  colony,  is  a  pure  culture  of  a  particular  species.  In  this  ex- 
periment we  are  more  apt  to  have  colonies  of  mould  f  uiigi  than  of 
bacteria,  but  the  principle  is  the  same,  viz. ,  that  a  colony  developed 
fn>ni  a  single  i^rm  is  a  pure  culture.  By  touching  our  platinum 


CULTURES   IN   SOLID   MEDIA.  71 

needle,  then,  to  such  a  colony,  which  is  quite  independent  of,  and 
well  separated  from,  all  others,  we  may  make  a  stick  culture  in  gela- 
tin or  agar,  and  preserve  the  pure  culture  for  further  study.  This 
is  a  most  important  advantage  which  pertains  to  the  use  of  solid 
culture  media.  It  is  a  singular  fact  that,  as  a  rule,  colonies  of  bac- 
teria which  lie  near  each  other  do  not  grow  together,  but  each  re- 
mains distinct.  If  there  are  but  few  colonies,  each  one,  haying 
plenty  of  room,  may  grow  to  considerable  size  ;  if  there  are  many 
and  they  are  crowded,  they  remain  small,  but  are  still  independent 
colonies. 

Now,  these  colonies  differ  greatly  in  their  appearance  and  char- 
acters of  growth,  according  to  the  species  (Fig.  43).  Some  are 
spherical,  and  these  may  be  translucent  or  opaque,  or  they  may  have 
an  opaque  nucleus  surrounded  by  a  transparent  zone.  Again,  the 


Fro.  43.— Colonies  of  Bacteria. 


outlines  may  be  irregular,  giving  rise  to  amoeba-like  forms,  or  to  a 
fringed  or  plaited  margin,  or  the  form  may  be  that  of  a  rosette,  etc. ; 
or  the  colony  may  appear  to  be  made  up  of  overlapping  scales  or 
masses,  or  of  tangled  filaments;  or  it  may  present  a  branching 
growth.  In  the  case  of  liquefying  bacteria,  when  the  colonies  have 
developed  in  a  gelatin  medium  they  commonly  do  not  at  once  cause 
liquefaction  of  the  gelatin,  but  at  the  end  of  twenty-four  hours  or 
more  the  gelatin  about  them  commences  to  liquefy  and  they  are 
seen  in  a  little  funnel  of  transparent  liquefied  gelatin ;  or  in  other 
cases  little  opaque  drops  of  liquefied  gelatin  are  seen,  which,  as  the 
liquefaction  extends,  run  together.  All  of  these  characters  are  best 
studied  under  a  low-power  lens,  with  an  amplification  of  five  to 
twenty  diameters  ;  and  by  a  careful  observation  of  the  differences  in 
the  form  and  development  of  colonies  we  are  greatly  assisted  in  the 
differentiation  of  species. 

Single,  isolated  colonies  do  not  always  contain  a  single  species, 
for  they  are  not  always  developed  from  a  single  cell.    We  may  have 


72  CULTURES   IN   SOLID   MEDIA. 

deposited  upon  our  plate,  exposed  as  above  described,  a  little  mass 
of  organic  material  containing  two  or  more  different  bacteria,  and 
this  would  serve  as  the  nucleus  of  a  colony  from  which  we  could  not 
obtain  a  pure  culture. 

Koch's  Plate  Method.— In  the  experiment  above  described, 
colonies  were  obtained  from  air-borne  germs  which  were  deposited 
upon  the  surface  of  our  gelatin  medium.  By  Koch's  famous  "  plate 
method  "  we  obtain  colonies  of  any  particular  microorganism  which 
we  desire  to  study,  or  of  two  or  more  associated  bacteria  which  we 
desire  to  study  separately  in  pure  cultures.  Evidently,  when  we 
have  obtained  separate  colonies  of  different  bacteria  upon  the  sur- 
face of  a  solid  culture  medium,  we  can  easily  obtain  a  pure  culture 
of  each  by  inoculating  stick  cultures  from  single  colonies. 

To  obtain  separate  colonies  we  resort  to  the  ingenious  method  of 
Koch.  Three  test  tubes  containing  a  small  quantity  of  nutrient 
gelatin  (or  of  agar)  are  commonly  employed.  The  tubes  are  num- 
bered 1,  2,  and  3.  The  first  step  consists  in  liquefying  the  nutrient 
jelly  by  heat,  and  it  will  be  well  for  beginners  to  place  the  tubes  in 
a  water  bath  having  a  temperature  of  about  40°  C.  (104°  F.)  for  the 
purpose  of  keeping  the  culture  material  liquid,  and  at  the  same  time 
at  a  temperature  which  is  not  high  enough  to  destroy  the  vitality  of 
the  bacteria  which  are  to  be  planted.  We  next,  by  means  of  a 
platinum- wire  loop  or  the  platinum  needle  used  for  stick  cultures, 
introduce  into  tube  No.  1  a  small  amount  of  the  culture,  or  material 
from  any  source,  containing  the  bacteria  under  investigation.  Care 
must  be  taken  not  to  introduce  too  much  of  this  material,  and  it 
must  be  remembered  that  the  smallest  visible  amount  may  contain 
many  millions  of  bacteria.  The  reason  for  using  three  tubes  will 
now  be  apparent.  It  is  usually  impossible  to  introduce  a  few  bac- 
teria into  tube  No.  1,  but  we  effect  our  object  by  dilution,  as  follows  : 
With  the  platinum- wire  loop  we  take  up  a  minute  drop  of  the  fluid  in 
tube  No.  1,  through  which  the  bacteria  have  been  distributed  by 
stirring,  and  carry  it  over  to  tube  No.  2.  Washing  off  the  drop  by 
stirring,  we  may  repeat  this  a  second  or  third  time — this  is  a  matter 
of  judgment  and  experience ;  often  it  will  suffice  to  carry  over  a 
single  ose  (the  German  name  for  the  platinum- wire  loop).  Next 
we  carry  over  one,  or  two,  or  three  ose  from  tube  No.  2  to  tube  No. 
3.  By  this  procedure  we  commonly  succeed  in  so  reducing  the  num- 
ber of  bacteria  in  tube  No.  3  that  only  a  few  colonies  will  develop 
upon  the  plate  which  we  subsequently  make  from  it;  or  it  may  happen 
that  the  dilution  has  been  carried  too  far  and  that  no  colonies  de- 
velop upon  the  plate  made  from  this  tube,  in  which  case  we  are 
likely  to  get  what  we  want  from  tube  No.  2.  The  next  step  is  to 
pour  the  liquid  gelatin  upon  sterilized  glass  plates,  which  are  num- 


CULTURES   IN   SOLID   MEDIA.  73 

bered  to  correspond  with  the  tubes.  The  plates  used  by  Koch  are 
from  eight  to  ten  centimetres  wide  and  ten  to  twelve  centimetres 
long.  They  must  be  carefully  cleaned  and  sterilized  in  the  hot-air 
oven,  at  150°  C.,  for  two  hours.  They  may  be  wrapped  in  paper  be- 
fore sterilization,  or  placed  in  a  metal  box  especially  made  for  the 
purpose.  In  order  that  the  liquid  gelatin  may  be  evenly  distributed 
upon  the  plate  the  apparatus  shown  in  Fig.  44  is  used.  This  con- 
sists of  a  glass  plate,  g,  supported  by  a  tripod  having  adjustable  feet. 
By  means  of  the  spirit  level  /  the  glass  plate  is  adjusted  to  a  hori- 
zontal position.  A  sterilized  glass  plate  is  placed  in  the  glass  tray, 
shown  in  the  figure,  and  the  gelatin  from  one  of  the  tubes  is  care- 
fully poured  upon  it  and  distributed  upon  its  surface  with  a  steril- 
ized glass  rod,  care  being  taken  not  to  bring  it  too  near  the  edge  of 
the  plate.  The  glass  tray  in  then  covered  until  the  gelatin  has 
cooled  sufficiently  to  become  solid,  after  which  plate  No.  1  is  re- 
moved and  plates  Nos.  2  and  3  are  made  in  the  same  way.  In 


Fio.44. 

order  to  save  time  it  is  customary  to  fill  the  glass  tray  shown  in  the 
figure  with  ice  water,  to  place  a  second  glass  support  upon  it,  and 
upon  this  the  sterilized  glass  plate  upon  which  the  liquid  gelatin  is 
poured.  This  is  protected  by  a  glass  cover,  as  before,  until  the  gela- 
tin becomes  solid. 

The  three  plates,  prepared  as  directed,  are  put  aside  in  a  glass 
jar  of  the  form  shown  in  Fig.  44,  one  being  supported  above  the 
pther  by  a  bench  of  sheet  zinc  or  glass. 

Petri's  Dishes. — A  modification  of  the  plate  method  of  Koch, 
which  has  some  advantages,  consists  in  the  use  of  three  small  glass 
dishes  of  the  same  form  as  the  larger  one  used  by  Koch  to  contain 
the  plates.  These  dishes  of  Petri  are  about  ten  to  twelve  centime- 
tres in  diameter  and  one  to  1.5  centimetres  high,  the  cover  being  of 
the  same  form  as  the  dish  into  which  the  gelatin  is  poured.  These 
dishes  take  less  room  in  the  incubating  oven  than  the  larger  glass 
jar  used  in  the  plate  method,  and  they  do  not  require  the  use  of  a 
levelling  apparatus.  The  colonies  also  may  be  examined  and 
counted,  if  desired,  without  removing  the  cover,  and  consequently 


74  CULTURES   IN  SOLID   MEDIA. 

without  the  exposure  which  occurs  when  a  plate  prepared  by  Koch's 
method  is  under  examination. 

In  agar-agar  cultures  or  in  gelatin  cultures  of  non-liquefying 
bacteria  made  in  Petri's  dishes,  we  may  examine  and  count  colonies, 
without  removing  the  cover,  by  inverting  the  dish. 

In  pouring  the  liquefied  gelatin  from  the  test  tubes  in  which  the 
dilution  has  been  made  into  sterilized  Petri's  dishes,  care  must  be 
taken  to  first  sterilize  the  lip  of  the  test  tube  by  passing  it  through 
the  flame  of  a  lamp.  We  may  at  the  same  time  burn  off  the  top  of 
the  cotton  plug,  then  remove  the  remaining  portion  with  forceps, 
when  the  lip  has  cooled,  for  the  purpose  of  pouring  the  liquid  into  the 
shallow  dish. 

Von  Esmarch' s  Roll  Tubes. — Another  very  useful  modification 
of  Koch's  plate  method  is  that  of  von  Esmarch.  Instead  of  pouring 
the  liquefied  gelatin  or  agar  medium  upon  plates  or  in  shallow 


FIG.  45. 

dishes,  it  is  distributed  in  a  thin  layer  upon  the  walls  of  the  test  tube 
containing  it.  This  is  done  by  rotating  the  tube  upon  a  block  of  ice 
or  in  iced  water.  Esmarch  first  used  a  tray  containing  iced  water, 
and  to  prevent  the  wetting  of  the  cotton  filter  a  cap  of  thin  rubber 
was  placed  over  the  end  of  the  tube.  It  is  more  convenient  to  turn 
the  tubes  upon  a  block  of  ice  having  a  horizontal  flat  surface,  in 
which  a  shallow  groove  is  first  made  by  means  of  a  test  tube  con- 
taining hot  water  (Fig.  45).  Or,  in  the  winter,  we  may  turn  the 
tube  under  a  stream  of  cold  water  from  the  city  supply — i.e.,  from  a 
faucet  in  the  laboratory.  A  little  practice  will  enable  the  student  to 
distribute  the  culture  medium  in  a  uniform  layer  on  the  walls  of  the 
test  tube,  and  as  soon  as  it  is  quite  solidified  these  may  be  placed 
a>i«lr  for  tin-  development  of  colonies  from  the  bacteria  which  had 
l>eeii  introduced.  When  roll  tubes  are  made  from  the  agar  jelly  it  is 
l»est  to  place  the  tubes  in  a  nearly  horizontal  position,  for  if  placed 
upright  at  once  the  film  of  jelly  is  likely  to  slip  from  the  walls  of  the 


CULTURES  IN  SOLID   MEDIA.  75 

tube.  This  is  due  to  the  fact  that  a  little  fluid  is  pressed  out  of  the 
jelly,  probably  by  a  slight  contraction  while  cooling.  If  the  tubes 
are  slightly  inclined  from  the  horizontal  the  film  does  not  slip  and 
the  fluid  accumulates  at  the  bottom.  After  a  day  or  two  they  may 
be  placed  in  an  upright  position. 

These  roll  tubes  possess  several  advantages.  They  are  quickly 
made  and  take  but  little  space  in  the  incubating  oven,  and  the  film 
of  jelly  is  protected  from  contamination  by  atmospheric  germs. 
When  colonies  have  formed  we  may  examine  them  through  the  thin 
walls  of  the  tube,  either  with  a  pocket  lens  or  a  low-power  objective. 
In  making  a  stick  culture  from  a  single  colony  in  one  of  these  roll 
tubes,  we  invert  the  tube,  remove  the  cotton  air  filter,  and  pass  the 
point  of  a  sterilized  platinum  needle  up  to  the  selected  colony.  In 
the  same  way  we  obtain  material  for  microscopical  examination. 

Streak  Cultures. — In  his  earlier  experiments  with  solid  culture 
media  Koch  made  "  streak  cultures"  by  drawing  the  point  of  a  plati- 
num needle,  charged  with  bacteria,  over  the  surface  of  a  gelatin  or 
agar  plate  ;  and  this  method  is  still  useful  in  certain  cases.  If  we 
draw  the  needle  over  the  moist  surface  several  times  in  succession 
the  greater  number  of  bacteria  will  be  deposited  in  the  first  streak, 
and  in  the  second  or  third  single  cells  are  likely  to  be  left  at  such 
intervals  from  each  other  that  each  will  develop  an  independent 
colony.  If  the  streaks  were  made  with  impure  stock  we  may  thus 
succeed  in  getting  separate  colonies  of  the  several  bacteria  contained 
in  it,  so  that  this  method  may  be  employed  for  obtaining  pure  cul- 
tures. But  for  this  purpose  it  is  much  inferior  to  the  plate  method, 
and  it  is  chiefly  used  for  observing  the  growth  of  bacteria  on  the  sur- 
face of  solid  culture  media.  Thus  we  commonly  make  a  streak  upon 
the  surface  of  cooked  potato  or  solidified  blood  serum  in  studying  the 
development  of  various  bacteria  on  these  culture  media. 

Cultures  upon  Blood  Serum. — The  use  of  blood  serum  as  a 
solid  medium  is  practically  restricted  to  stick  cultures  and  streak 
cultures,  for  we  cannot  substitute  it  for  the  gelatin  and  agar  media 
in  making  plates  and  roll  tubes.  This  is  because  it  only  becomes  solid 
at  a  temperature  which  would  be  fatal  to  most  bacteria  (70°  C.),  and 
when  once  made  solid  by  heat  cannot  again  be  liquefied.  Its  use  is, 
therefore,  restricted  mainly  to  the  cultivation  of  bacteria  for  which 
it  is  an  especially  favorable  medium.  It  may  be  used,  however,  in 
combination  with  a  gelatin  or  agar  medium.  For  this  purpose  it  is 
most  conveniently  kept  in  a  fluid  condition  in  the  little  flasks  hereto- 
fore described  ("  Sternberg's  bulbs  "). 

The  gelatin  or  agar  jelly  in  test  tubes  is  liquefied  by  heat  and 
cooled  in  a  water  bath  to  about  40°  C.  The  desired  amount  of  ste- 
rile blood  serum  is  then  forced  into  each  tube  by  passing  the  slender 


76  CULTURES   IN   SOLID   MEDIA. 

neck  of  the  little  flask  along  the  side  of  the  cotton  filter  (see  Fig.  46) 
and  applying  gentle  heat  to  the  bulb.  The  slender  neck  is  first  ste- 
rilized by  passing  it  through  a  flame,  and  the  point  is  broken  off 
with  sterile  forceps.  After  inoculating  the  liquefied  medium  in  the 
test  tubes  in  the  usual  manner  we  may  make  plates  or  roll  tubes. 

Cultures  on  Cooked  Potato.— The  method  of  preparing  pota- 
toes for  surface  cultures  has  already  been  given  (page  48).  It  was 
in  using  them  that  Koch  first  got  his  idea  of  the  importance  of  solid 
media,  which  led  to  his  introduction  of  the  use  of  gelatin  and  agar- 
agar  and  the  invention  of  the  plate  method.  By  means  of  streak 


FIG.  46. 

cultures  upon  potato  he  had  succeeded  in  obtaining  isolated  colonies 
and  pure  cultures.  We  now  use  the  potato  chiefly  for  the  purpose 
of  differentiating  species.  Some  bacteria  grow  on  the  surface  of 
cooked  potato  and  some  do  not.  Those  which  do  present  various 
characters  of  growth.  Thus  we  have  differences  as  to  color,  as  to 
rapidity  of  growth,  as  to  the  character  of  the  mass  formed — thick 
or  thin,  viscid,  moist  or  dry,  restricted  to  line  of  inoculation  or  ex- 
tending over  the  entire  surface,  etc. 

Instead  of  using  a  cut  section  of  the  potato  in  the  manner  here- 
tofore described,  we  may  make  a  puree  by  mashing  the  peeled  and 
cooked  tubers  and  distributing  the  mass  in  Erlenmeyer  flasks.  After 


CULTURES   IN   SOLID   MEDIA.  77 

thorough  sterilization  by  steam  the  culture  medium  is  ready  for  use. 
In  the  same  way  other  vegetables,  or  bread,  etc. ,  may  be  used  for 
special  purposes,  and  especially  for  cultures  of  the  mould  fungi. 

Potatoes  usually  have  a  slightly  acid  reaction,  and  on  this  ac- 
count certain  bacteria  will  not  grow  upon  them.  This  acid  reaction 
is  not  constant  and  differs  in  degree,  and  as  a  result  we  may  have 
decided  differences  in  the  growth  of  the  same  species  upon  different 
potatoes.  To  overcome  this  objection  the  writer  has  sometimes  neu- 
tralized the  cones  of  potato  in  test  tubes  (see  Fig.  21,  page  48)  by 
first  boiling  them  in  water  containing  a  little  carbonate  of  soda. 
The  liquid  is  poured  off  after  they  have  been  in  the  steam  sterilizer 
for  half  an  hour,  and  they  are  returned  for  sterilization. 

Salomonson's  Method  of  cultivation  in  capillary  tubes  has  a  his- 
torical value  only  since  the  introduction  of  Koch's  plate  method. 

The  following  modifications  of  Koch's  plate  cultures  have  recently 
been  introduced : 

Kruse  (1894)  pours  the  liquefied  gelatin  or  agar  into  Petri  dishes, 
and  after  it  is  solidified  brushes  the  surface  with  a  sterilized  camel's- 
hair  brush  which  has  been  dipped  into  water  containing  in  suspen- 
sion—properly diluted— the  bacteria  to  be  studied.  By  this  procedure 
surface  colonies  only  are  obtained.  Von  Freudenreich  (1894)  prefers 
to  pour  the  contents  of  the  test  tube  upon  the  surface  of  the  sterile 
medium,  in  Petri  dishes.  The  fluid  is  allowed  to  run  off  by  placing 
the  Petri  dish  in  a  vertical  position,  and  this  is  subsequently  placed  in 
the  incubating  oven  in  an  inverted  position — i.e.,  with  cover  below. 
To  obtain  satisfactory  plates  with  well-separated,  superficial  colonies 
it  may  be  necessary  to  use  two  or  three  dilutions,  made  in  sterilized 
water  in  the  usual  way — i.e.,  from  one  tube  to  another,  by  means  of 
the  platinum  wire  having  a  loop  at  its  extremity. 


IX. 


CULTIVATION  OF  ANAEROBIC  BACTERIA. 

PASTEUR  (1861)  first  pointed  out  the  fact  that  certain  species  of 
bacteria  not  only  grow  in  the  entire  absence  of  oxygen,  but  that  for 
some  no  growth  can  occur  in  the  presence  of  this  gas.  Such  bacteria 
are  found  in  the  soil,  and  in  the  intestines  of  man  and  the  lower  ani- 
mals. The  cultivation  of  "strict  anaerobics"  calls  for  methods  by 
which  oxygen  is  excluded.  The  "facultative  anaerobics ''  grow 


Fio.  47. 


FIG. 48. 


either  in  the  presence  or  absence  of  oxygen.  There  are  various  gra- 
dations in  this  regard,  from  the  strictly  aerobic  species  which  re- 
•  piiro  an  abundance  of  oxygen  and  will  not  grow  in  its  absence,  to 
the  strictly  anaerobic  species  which  will  not  grow  if  there  is  a  trace 
of  oxygen  in  the  medium  in  which  we  propose  to  cultivate  them. 
Among  the  most  interesting  pathogenic  bacteria  which  are  strictly 
anaerobic  are  the  bacillus  of  tetanus,  the  bacillus  of  malignant 
and  tin-  ku-illus  of  symptomatic  anthrax* 


CULTIVATION   OF   ANAEROBIC  BACTERIA.  79 

If  we  make  an  inoculation  of  one  of  the  species  which  is  not 
strictly  anaerobic  into  a  test  tube  containing  nutrient  gelatin  or  agar- 
agar,  we  may  have  a  development  all  along  the  line  of  puncture, 
and  this  may  be  more  abundant  below,  as  in  Fig.  47.  But  when  we 
make  a  long  stick  culture  with  a  strict  anaerobic  the  development 
occurs  only  near  the  bottom  of  the  line  of  puncture  (Fig.  48). 

We  may  then,  if  we  have  a  pure  culture  to  start  with,  propagate 
these  anaerobic  bacilli  in  long  stick  cultures.  It  is  best  to  use  tubes 
which  have  been  recently  sterilized,  as  boiling  expels  the  air  from 
the  culture  medium ;  and  a  very  slender  needle  should  be  used  in 
making  the  inoculation.  To  prevent  the  absorption  of  oxygen  a 
layer  of  sterilized  olive  oil  may  be  poured  into  the  tube  after  the  in- 
oculating puncture  has  been  made,  or  it  may  be  filled  up  with  agar 
jelly  which  has  been  cooled  to  about  40°  C.  Roux  has  proposed  to 
prevent  the  absorption  of  oxygen  by  the  culture  medium  by  plant- 
ing an  aerobic  bacterium — Bacillus  subtilis — upon  the  surface,  after 
making  a  long  stick  culture  with  the  anaerobic  species.  The  agar 
jelly  is  first  boiled  and  quickly  cooled  ;  the  inoculation  is  then  made 
with  a  slender  glass  needle  ;  some  sterile  agar  cooled  to  40°  C.  is 
poured  into  the  tube,  and  when  this  is  solid  the  aerobic  species  is 
planted  upon  the  surface.  The  top  of  the  test  tube  is  then  closed 
hermetically  and  it  is  placed  in  the  incubating  oven.  The  aerobic 
species  exhausts  the  oxygen  in  the  upper  part  of  the  tube  by  its 
growth  on  the  surface  of  the  culture  medium,  and  the  anaerobic 
species  grows  at  the  bottom  of  the  tube.  To  obtain  material  for  a 
new  culture  or  for  microscopical  examination  the  test  tube  is  broken 
near  its  bottom. 

Cultures  in  liquid  media  may  be  made  by  exhausting  the  air  in 
a  suitable  receptacle  or  by  displacing  it  with  hydrogen  gas.  The 
first-mentioned  method  has  been  largely  used  in  Pasteur's  laboratory, 
but  methods  in  which  hydrogen  gas  takes  the  place  of  atmospheric 
air  in  the  culture  tube  are  more  easily  applied  and  require  simpler 
apparatus.  The  flask  shown  in  Fig.  49  may  be  used  in  connection 
with  an  air  pump.  The  sterile  culture  liquid  is  first  introduced  into 
a  long-necked  flask  and  inoculated  with  the  anaerobic  bacillus  to  be 
cultivated.  The  neck  of  the  flask  is  then  drawn  out  in  a  flame  at  c. 
The  open  end  is  then  connected  with  a  Sprengle's  pump  or  some 
other  apparatus  for  exhausting  the  air.  The  flask  is  placed  in  a 
water  bath  at  40°  C. ,  which  causes  ebullition  at  the  diminished  pres- 
sure, and  the  exhaustion  is  continued  for  about  half  an  hour.  The 
narrow  neck  is  then  sealed  at  c  by  the  use  of  a  blowpipe  flame. 

The  flask  shown  in  Fig.  49,  which  can  be  made  from  a  test  tube, 
may  also  be  used  in  connection  with  a  hydrogen  apparatus.  In  this 
case  a  slender  glass  tube  is  passed  into  the  flask,  as  shown  in  Fig. 


80 


CULTIVATION   OF   ANAEROBIC   BACTERIA. 


50,  and  this  is  connected  with  a  hydrogen  apparatus  by  a  rubber 
tube,  The  hydrogen  is  allowed  to  bubble  through  the  culture 
liquid  in  a  full  stream  for,  ten  to  fifteen  minutes,  in  order  that  all  of 
the  oxygen  in  the  flask  may  be  removed  by  displacement.  Then. 
while  the  gas  is  still  flowing,  the  flask  is  sealed  at  a  with  a  blow- 
pipe flame,  the  hydrogen  tube  being  left  in  position  and  melted  fast 
to  the  flask.  Some  little  #kill  is  required  in  the  successful  perform- 
ance of  the  last  step  in  this  procedure,  and  it  will  be  easier  for  those 


Fio.  49. 


TIG.  51. 


who  are  not  skilful  in  the  use  of  the  blowpipe  to  use  Salomonson's 
tube,  shown  in  Fig.  51.  In  this,  hydrogen  is  admitted  through  the 
arm  b,  and  escapes  through  the  cotton  plug  a.  The  vertical  tube  is 
sealed  at  c  while  the^as  is  flowing,  and  then  the  horizontal  tube  at  b. 
/•V///AW*  Method. — Instead  of  these  tubes  specially  made  for 
the  purpose,  an  ordinary  test  tube  may  be  used,  as  recommended  by 
Krankel.  This  is  closed  by  a  soft  rubber  cork  through  which  two 
Ljlass  tuhes  pass— one,  reaching  nearly  to  the  bottom  of  the  test  tube, 


CULTIVATION   OF   ANAEROBIC   BACTERIA. 


81 


for  the  admission  of  hydrogen,  which  passes  through  the  liquefied 
culture  medium  ;  and  the  other  a  short  tube  for  the  escape  of  the  gas. 
The  outlet  tube  is  sealed  in  the  flame  of  a  lamp  while  the  gas  is 
freely  flowing,  and  after  sufficient  time  has  elapsed  to  insure  the 
complete  expulsion  of  atmospheric  oxygen — which,  when  the  hydro- 
gen flows  freely,  requires  about  four  minutes  (Frankel) — melted 
paraffin  is  applied  freely  to  the  rubber  stopper  to  prevent  leakage  of 
the  hydrogen  and  entrance  of  oxygen.  A  roll  tube  may  then  be 
made  after  the  manner  of  Esmarch,  and,  after  colonies  have  de- 
veloped, the  anaerobic  culture  will  appear  as  shown  in  Fig.  52. 

To  isolate  anaerobic  bacteria  in  pure  cultures  it  is  well  to  make  a 


FIG.  58.  FIG.  53. 

series  of  dilutions  as  heretofore  described  for  aerobic  cultures  ;  we 
will  then  usually  obtain  isolated  colonies  in  tube  No.  2  or  No.  3  of  a 
series,  and  by  removing  the  rubber  stopper  we  may  transplant  bac- 
teria from  these  colonies  to  deep  stick  cultures  in  nutrient  gelatin  or 
agar. 

The  Writer's  Method. — The  following  simple  method  has  been 
successfully  employed  by  the  writer: 

Three  Esmarch  roll  tubes  are  prepared  as  is  usual  for  aerobic  cul- 
tures. The  cotton  air  filter,  or  a  portion  of  it,  is  then  pushed  down 
the  tubes  for  a  short  distance,  as  shown  at  a,  Fig.  53.  A  section  of 
a  soft  rubber  stopper  carrying  two  glass  tubes  is  then  pushed  into  the 
6 


82 


CULTIVATION  OF  ANAEROBIC   BACTERIA. 


test  tube  for  about  half  an  inch,  as  shown  at  b,  Fig.  53.  The  space 
above  the  cork  is  then  filled  with  melted  sealing  wax,  which  I  have 
found  to  prevent  leakage  better  than  paraffin,  which  contracts  upon 
cooling.  The  test  tube  is  inverted  while  hydrogen  is  passed  through 
the  tube  c,  and  by  reason  of  its  levity  the  gas  quickly  passes  through 
the  cotton  air  filter  and  displaces  the  oxygen  in  the  test  tube  (Fig. 
54).  After  allowing  the  gas  to  flow  for  a  few  minutes  the  outlet 
tube  is  first  sealed  in  a  flame  and  then  the  inlet  tube.  As  the  cotton 
filter  is  interposed  between  the  rubber  stopper  and  the  culture  mate- 
rial, no  special  precautions  need  be  taken  for  the  sterilization  of  the 
rubber  cork  and  the  glass  tubes  which  it  carries. 


Fio.  54. 


FIG.  55. 


This  method  is  more  convenient  than  that  previously  described, 
and  the  only  objection  to  it  is  that  the  oxygen  is  not  completely  re- 
moved from  the  film  of  solid  gelatin  or  agar  attached  to  the  walls  of 
the  test  tube.  But  by  passing  the  hydrogen  for  a  long  time  it  would 
seem  that  by  diffusion  the  oxygen  remaining  in  this  thin  layer 
would  be  gotten  rid  of.  At  all  events,  this  method  will  serve  for  all 
except  the  very  strict  anaerobics. 

Method  of  Esmarch.—ThQ  following  method  has  been  proposed 
by  Esmarch  :  Three  roll  tubes  are  made  in  the  usual  way,  and  into 
these  liquid  gelatin,  that  is  nearly  cooled  to  the  point  of  becoming 
solid,  is  poured.  This  fills  the  tube  without  melting  the  layer  of 


CULTIVATION  OP  ANAEROBIC  BACTERIA. 


83 


gelatin,  previously  cooled  upon  its  walls,  which  contains  the  bacteria 
under  investigation.  When  the  anaerobic  colonies  have  developed 
the  test  tube  must  be  broken  to  get  at  them,  or  the  cylinder  of  gela- 
tin may  be  removed  by  first  warming  the  walls  of  the  tube. 

Another  method,  recommended  by  Liborius,  consists  in  distri- 
buting the  bacteria  in  test  tubes  nearly  filled  with  nutrient  gelatin  or 
agar  which  has  been  recently  boiled  to  expel  air.  Colonies  of  anaero- 
bic bacteria  will  develop  near  the  bottom  of  such  a  tube,  while  the 
aerobic  species  will  only  grow  near  the  surface.  The  cylinder  of 
jelly  is  removed  by  heating  the  walls  of  the  tube,  and  sections  are 


}  a 


FIG.  56. 

made  with  a  sterilized  knife  for  the  purpose  of  "obtaining  material 
from  individual  colonies  for  further  cultures,  etc. 

Koch  and  his  pupils  are  in  the  habit  of  testing  the  aerobic  char- 
acter of  bacteria  in  plate  cultures  by  covering  the  recently  made 
plates  with  a  thin  sheet  of  mica  which  has  been  sterilized  by  heat. 
The  strictly  aerobic  species  do  not  grow  under  such  a  plate  ;  but, 
according  to  Liborius,  the  exclusion  of  oxygen  is  not  sufficiently 
complete  for  the  growth  of  strict  anaerobics. 

Buchner's  Method  consists  in  the  removal  of  oxygen  by  means 
of  pyrogallic  acid.  The  anaerobic  species  under  investigation  is 
planted  in  recently  boiled  agar  jelly  in  a  small  test  tube.  This  is 
placed  in  a  larger  tube  having  a  tightly  fitting  rubber  stopper,  as 
shown  in  Fig.  55.  The  small  tube  is  supported  by  a  bent-wire 


84 


CULTIVATION  OF  ANAEROBIC  BACTERIA. 


stand,  and  in  the  lower  part  of  the  large  tube  are  placed  ten  cubic 
centimetres  of  a  ten-per-cent  solution  of  caustic  potash,  to  which  one 
gramme  of  pyrogallic  acid  is  added.  The  absorption  of  the  oxygen 
takes  some  time,  but,  according  to  Buchner,  it  is  finally  so  complete 
that  strict  anaerobics  grow  in  the  small  tube. 

In  practice,  cultivation  in  an  atmosphere  of  hydrogen  will  be 
found  the  most  convenient  method,  and  for  this  any  form  of  hydro- 
gen generator  may  be  used.  The  writer  is  in  the  habit  of  using  the 
form  shown  in  Fig.  56.  A  perforation  a  quarter  of  an  inch  in 
diameter  is  drilled  through  the  bottom  of  a  wide-mouthed  bottle. 
Some  fragments  of  broken  glass  are  then  put  into  the  bottle,  f orm- 


FIQ.  57. 

ing  a  layer  two  or  three  inches  thick.  Upon  this  is  placed  a  quan- 
tity of  granulated  zinc.  This  bottle  has  a  tightly  fitting  cork, 
through  which  passes  a  metal  tube  having  a  stopcock.  The  bottle 
is  placed  in  a  glass  jar  containing  diluted  sulphuric  acid  (one  part 
by  weight  of  sulphuric  acid  to  eight  parts  of  water).  The  acid,  ris- 
ing through  the  perforation  in  the  bottom  of  the  bottle,  when  it 
comes  in  contact  with  the  zinc  gives  rise  to  an  abundant  evolution 
•  •I  hydrogen,  which  escapes  by  the  tube  a  when  the  stopcock  is 
open.  When  this  is  closed  the  gas  forces  the  acid  back  from  con- 
tact with  the  zinc.  To  remove  any  trace  of  oxygen  present  the 
#as  may  be  passed  through  a  solution  of  pyrogallic  acid  in  caustic 
potash. 

Evidently  plates  prepared   by  Koch's  method,  or  Esmarch  roll 


CULTIVATION   OF  ANAEROBIC   BACTERIA.  85 

tubes,  may  be  placed  in  a  suitable  receiver  and  the  air  exhausted,  or 
hydrogen  substituted  for  atmospheric  air.  Such  an  apparatus  for 
hydrogen  has  been  devised  by  Blucher  and  is  shown  in  Fig.  57.  A 
glass  dish,  A,  contains  a  smaller  dish,  B,  which  has  a  diameter  of 
about  seven  centimetres.  The  small  dish  is  kept  in  its  position  in 
the  centre  of  the  larger  one  by  the  wire  ring,  having  three  project- 
ing arms,  which  is  shown  in  the  figure.  The  culture  medium  con- 
taining the  anaerobic  bacteria  to  be  cultivated  is  poured  into  the 
small  dish  and  the  glass  funnel  D  is  put  in  position.  This  is  held 
in  its  place  by  a  weight  of  lead  which  encircles  the  neck  of  the  fun- 
nel at  F.  A  mixture  of  glycerin  and  water  (twenty  to  twenty -five 
per  cent)  is  poured  into  the  dish  A  to  serve  as  a  valve  to  shut  off 
the  atmospheric  air  from  the  interior  of  the  funnel  D.  Hydrogen 
gas  is  introduced  through  the  tube  E,  which  is  connected  by  a  rub- 
ber tube  with  a  hydrogen  apparatus. 

A  somewhat  similar  apparatus  has  been  devised  by  Botkin,  in 
which  the  hydrogen  is  admitted  beneath  a  bell  jar  covering  small 
glass  dishes  containing  the  culture  medium.  We  believe  that  in 
practice  the  writer's  method  (page  81),  in  which  Esmarch  roll  tubes 
are  first  made,  will  be  found  more  convenient  than  either  of  the  last- 
mentioned  methods  of  preserving  plates  in  an  atmosphere  of  hydro- 
gen ;  or  roll  tubes  may  be  prepared  in  the  way  usually  practised  in 
cultivating  aerobic  bacteria,  and  these  may  be  placed  in  a  suitable 
receptacle  which  can  be  filled  with  hydrogen. 


X. 
INCUBATING  OVENS  AND  THERMO-REGULATORS. 

THE  saprophytic  bacteria  generally,  and  many  of  the  pathogenic 
species,  grow  at  the  ordinary  temperature  of  occupied  apartments 
(20°  to  25°  C.)  ;  but  some  pathogenic  species  can  only  be  cultivated 
at  a  higher  temperature,  and  many  of  those  which  grow  at  the 
"  room  temperature "  develop  more  rapidly  and  vigorously  when 
kept  in  an  incubating  oven  at  a  temperature  of  35°  to  38°  C.  Every 
bacteriological  laboratory  should  therefore  be  provided  with  one  or 
more  brood  ovens  provided  with  thermo-regulators  to  maintain  a 
constant  temperature.  These  incubating  ovens  are  made  with  dou- 
ble walls  surrounding  an  air  chamber.  The  space  between  the  dou- 
ble walls  is  filled  with  water,  which  is  usually  heated  by  a  small  gas 
flame.  The  gas  passes  through  the  thermo-regulator,  and  its  flow 
is  automatically  controlled  for  any  temperature  to  which  this  is  ad- 
justed. The  exterior  of  the  incubating  oven  is  covered  with  felt  or 
asbestos  to  prevent  the  loss  of  heat  by  radiation.  A  simple  and 
cheap  form  which  answers  every  purpose  is  shown  in  Fig.  58.  The 
quadrangular  box  with  double  walls  should  be  made  of  zinc  or  cop- 
per. An  outer  metal  door  covered  with  non-conducting  material, 
and  an  inner  door  of  glass,  give  access  to  the  interior  space  ;  and  a 
thermometer  introduced  through  an  aperture  in  the  top  (Fig.  58,  b) 
shows  the  temperature  of  this  space  when  the  door  is  closed.  The 
stopcock  e  permits  the  drawing  off  of  the  water  from  the  space  be- 
tween the  double  walls,  and  the  glass  tube  d  shows  the  height  of 
the  water,  as  it  is  connected  with  the  space  containing  it.  The 
thermo-regulator  passes  through  an  aperture  at  one  side  of  the  oven 
into  the  water,  the  temperature  of  which  controls  the  flow  of  gas. 

The  ordinary  thermo-regulator  is  shown  in  Fig.  59  as  manufac- 
tured by  Rohrbeck.  A  glass  receptacle,  shaped  like  an  ordinary 
test  tube,  has  an  arm,  c,  for  the  escape  of  the  gas,  which  enters  by 
the  bent  tube  a,  which  passes  through  a  perforated  cork  and  is  ad- 
justable up  and  down.  Tube  a  is  connected  with  the  gas  supply  and 
tube  c  with  the  burner  by  means  of  rubber  tubing.  A  glass  parti- 
extending  downward  as  a  tube,  </,  makes  an  enclosed  space  in 


INCUBATING   OVENS   AND   THERMO-REGULATORS. 


87 


the  lower  part  of  the  instrument,  and  this,  when  immersed  in  water, 
acts  as  a  thermometer  bulb.  This  space  contains  mercury  below 
and  air  or  the  vapor  of  ether  above.  When  the  air  is  expanded  by 
heat  the  mercury  is  forced  up  the  tube  g  until  it  meets  the  end  of 
the  inlet  tube  for  gas  at  h,  and  by  shutting  off  the  flow  of  gas  pre- 
vents the  temperature  from  going  any  higher.  A  small  opening  in 
the  inlet  tube  at  e  permits  a  small  amount  of  gas  to  flow,  so  that  the 
flame  under  the  brood  oven  ( Fig.  58,  /)  may  not  be  entirely  extin- 
guished. The  lower  end  of  the  bent  tube  a  is  bevelled,  so  that  a  tri- 
angular opening  is  formed,  which  is  closed  gradually  by  the  rising 


FIG.  58. 

mercury,  instead  of  abruptly  as  would  be  the  case  if  the  lower  end 
of  the  tube  a  were  cut  off  square.  To  adjust  the  temperature  in 
the  air  space  of  the  incubating  oven  when  the  thermo-regulator  is  in 
position,  a  full  flow  of  gas  is  admitted  to  the  burner  until  the  ther- 
mometer (Fig.  58,  b)  shows  the  desired  temperature ;  then  the  bent 
tube  a  is  pushed  down  through  the  cork  until  its  lower  extremity 
meets  the  mercury  and  the  flame  /  is  somewhat  reduced.  The  ap- 
paratus is  then  left  for  a  time,  to  see  whether  the  flame  runs  too  high 
or  too  low,  and  a  further  adjustment  is  made.  When  the  changes 
in  the  exterior  temperature  are  slight  and  the  gas  pressure  regular 
the  temperature  in  the  air  chamber  is  controlled  with  great  precision. 
But  this  is  not  the  case  under  the  reverse  conditions.  Changes  in 


88 


INCUBATING   OVENS   AND   THERMO-REGULATORS. 


the  pressure  of  gas,  especially,  interfere  with  the  maintenance  of  a 
constant  temperature,  and  for  this  reason  a  pressure  regulator  will 
be  required  when  great  precision  is  desired.  That  of  Moitessier  is 
commonly  used  in  bacteriological  laboratories  (Fig.  60).  But  for 
most  purposes  variations  of  temperature  of  1°  to  2°  C.  are  not  of 
great  importance.  For  ordinary  use  a  brood  oven  should  be  regu- 
lated to  about  35°  to  37°  C.  It  is  best  to  have  a  little  cylindrical 
screen  of  mica  around  the  gas  jet  beneath  the  incubating  oven,  for 
the  purpose  of  preventing  the  flame  from  being  extinguished  by  cur- 
rents of  air  (Fig.  61). 

Koch's  ingenious  automatic  device  for  shutting  off  the  gas  if  the 
flame  is  accidentally  extinguished  is  shown  in  Fig.  62. 


FIG.  59. 


FiO.60. 


Another  form  of  thermo-regulator,  which  answers  very  well,  is 
that  of  Reichert  (Fig.  63).  In  this  the  gas  enters  at  a  and  escapes 
at  c.  The  mercury,  which  fills  the  bulb,  shuts  off  the  gas  at  the 
point  for  which  the  instrument  is  regulated.  By  means  of  the 
screw  d  the  height  of  the  mercury  in  the  tube  may  be  very  accu- 
rately adjusted  for  any  desired  temperature. 

The  regulator  of  Bohr,  shown  in  Fig.  64,  is  more  sensitive  than 
that  of  Reichert,  and  rather  simpler  in  construction  than  the  usual 
form  shown  in  Fig.  59.  The  thermometer  bulb  a  contains  only  air, 
and  the  gas  which  passes  through  the  tube  /  is  shut  off  at  the 
proper  temperature  by  the  mercury  in  the  U-shaped  tube  c.  The 
stopcock  b  is  left  open  when  the  bulb  a  is  immersed  in  the  water 


INCUBATING   OVENS   AND   THERMO-REGULATORS. 


89 


bath,  and  when  the  proper  temperature  is  reached  is  closed  so  as  to 
confine  the  air  in  the  bulb.     An  increase  of  temperature  now  causes 


FIG.  61.  FIG.  62. 

the  air  in  the  bulb  to  expand,  the  mercury  in  the  U-tube  is  forced  up 


a,  ... 


.-d 


FIG.  63. 


FIG.  64. 


and  shuts  off  the  gas  flowing  through  the  tube  /  at  its  lower  ex- 
tremity,  d.     A  small  opening,  e,  permits  sufficient  gas  to  pass  to 


90 


I  NCI"  HATING   OVENS   AND    THERMO-REGULATORS. 


maintain  a  small  flame  which  must  not  be  sufficient  by  itself  to  keep 
up  the  desired  temperature  in  the  water  bath. 

Altmann  has  recently  (1891)  described  a  thermo-regulator  which 
is  made  by  Miincke,  of  Berlin,  and  which  is  shown  in  Fig.  65.  This 
is  >aid  to  act  with  great  precision.  It  is  a  modification  of  Reichert's 


FIG.  65. 


Fio.  66. 


regulator.     Its  mode  of  action  will  be  readily  understood  by  a  refe- 
rence to  the  figure. 

A  thermo-regulator  which  gives  very  accurate  results,  which  ,-nv 
not  influenced  by  differences  in  pressure,  is  that  invented  by  the 


Fio.  67. 


writer  over  twenty  years  ago.  The  regulating  thermometer  may 
""tain  mercury  only,  or  air  and  mercury,  as  shown  in  the  thermo- 
regulator  for  gas  (  Kifr  50).  In  the  simplest  form  a  large  bulb  con- 
aming  mercury  is  used,  and  a  platinum  wire  is  hermetically  sealed 
a  glass  so  as  to  have  contact  with  the  mercury  (Fig.  (5(1,  ft). 


INCUBATING   OVENS   AND    THERMO-REGULATORS. 


91 


Another  platinum  wire  passes  down  the  tube  of  the  thermometer,  b, 
and  is  adjustable  for  any  desired  temperature.  The  gas  passes 
through  a  valve  which  is  controlled  by  an  electro-magnet.  A 
simple  form  of  valve  is  shown  in  Fig.  67.  The  bent  tube  a  is  con- 
nected with  the  gas  supply  by  a  piece  of  rubber  tubing.  The  up- 
right arm  of  this  tube  is  enclosed  in  a  larger  tube,  fr,  having  an  out- 


Fio.  68.  FIG.  69. 

let,  e,  which  is  connected  with  the  burner  under  the  incubating 
oven.  The  upper  end  of  this  larger  tube  is  closed  by  means  of  a 
piece  of  sheet  rubber,  which  prevents  the  escape  of  gas.  When  this 
is  depressed  by  means  of  the  lever  c,  the  flow  of  gas  through  the 
valve  is  arrested.  The  lever  c  has  attached  to  it  the  armature  d, 
and  is  operated  by  an  electro-magnet  under  the  control  of  the  regu- 
lating thermometer. 


92  INCUBATING   OVENS   AND    THERMO-REGULATORS. 

When  the  thermometer  is  immersed  in  a  water  bath  the  tem- 
perature of  which  it  is  desired  to  regulate,  and  the  proper  electric 
connections  are  made,  it  acts  as  a  circuit  breaker.  When  the  de- 
sired temperature  is  reached  the  mercury  in  the  tube  of  the  ther- 
mometer touches  the  wire  b  (Fig.  66),  an  electric  circuit  is  com- 
pleted, and  the  valve  is  closed,  shutting  off  the  gas  supply  and 
preventing  the  temperature  from  going  any  higher.  When  contact 
is  broken  in  the  thermometer  tube  the  valve  opens  and  permits  the 
gas  to  flow  again.  A  small  opening,  o  (Fig.  67),  permits  the  con- 
stant flow  of  a  sufficient  amount  of  gas  to  prevent  the  flame  from 
being  extinguished.  In  practice,  however,  it  is  better  to  have  a 
small  side  jet  of  gas,  quite,  independent  of  that  which  passes  through 
the  valve,  which  burns  constantly  and  relights  the  principal  jet  when 


Fio.  70. 


the  valve  is  opened.  This  apparatus  is  very  well  adapted  for  regu- 
lating the  temperature  of  a  water  bath  with  precision,  but  for  gene- 
ra 1  use  in  collection  with  incubating  ovens  the  ordinary  gas  regu- 
lator is  preferable,  on  account  of  the  trouble  connected  with  keeping 
a  galvanic  battery  in  order  when  it  is  required  to  act  at  frequent 
intervals  "  on  a  closed  circuit,"  for  weeks  and  months  together. 

The  incubating  apparatus  of  D'Arsonval  is  shown  in  Fig.  68.  It 
is  a  cylindrical  vessel  of  copper  having  double  walls,  and  is  provided 
with  the  thermo-regulator  of  D'Arsonval,  by  which  very  accurate 
regulation  is  maintained  at  any  desired  temperature.  In  its  form 
this  apparatus  is  not  as  convenient  as  are  the  brood  ovens  made 
in  the  form  shown  in  Fig.  58,  with  a  swinging  door  which  givrs 
easy  access  to  the  interior,  which  is  provided  with  one  or  more 
shelves  upon  which  the  cultures  are  placed.  Various  modifications 


INCUBATING   OVENS   AND   THERMO-REGULATORS.  93 

of  this  simple  and  convenient  incubating  oven  are  manufactured 
by  Robrbeck  and  by  Muncke,  of  Berlin.  The  apparatus  of  D'Ar- 
sonval,  and  other  forms  in  favor  at  the  French  capital,  may  be  ob- 
tained from  Wiesnegg,  of  Paris.  The  last-named  manufacturer 
also  supplies  the  incubating  oven  and  thermo-regulator  described  by 
Roux  (1891).  This  is  shown  in  Fig.  69.  The  regulator  is  formed  of 
two  metallic  bars,  One  of  steel  and  the  other  of  zinc  ;  these  are 
soldered  together  in  the  shape  of  a  letter  U  ;  the  regulator  is  seen  in 
position  in  the  cut  (Fig.  69).  The  most  dilatable  metal  (zinc)  is  on 
the  outside.  When  the  temperature  is  raised  the  arms  of  the  U  ap- 
proach each  other,  and  the  reverse  when  it  falls.  The  method  by 
which  regulation  is  effected  is  shown  in  Fig.  70.  The  U-shaped 
regulator  is  placed  vertically,  and  one  of  its  branches,  A,  is  firmly 
fixed  to  the  wall  of  the  incubating  oven  ;  the  other,  free  arm  car- 
ries a  horizontal  bar  which  projects  through  the  wall  of  the  incu- 
bator in  an  opening  which  permits  it  to  move  freely  under  the  influ- 
ence of  a  change  in  the  temperature  within.  The  end  of  this 
projecting  bar  is  turned  up  at  a  right  angle  and  the  screw  p  passes 
through  it  ;  this  can  be  fixed  at  any  desired  point  by  means  of  the 
nut  e.  The  end  of  the  screw  p  rests  against  the  stem  of  a  conical 
brass  valve  which  controls  the  flow  of  gas.  The  valve  is  closed  by  a 
spiral  spring  and  opened  by  the  screw  p  under  the  control  of  the 
thermo-regulator. 

In  the  absence  of  gas  incubating  ovens  may  be  heated  by  a  small 
petroleum  lamp,  and  various  devices  have  been  invented  for  control- 
ling the  temperature.  Reichenbach  describes  an  apparatus  for  this 
purpose  in  the  Centralblatt  fiir  Bakteriologie,  Vol.  XV.,  p.  847, 
1894.  Dr.  Borden  of  the  U.  S.  Army  has  also  invented  a  thermo- 
regulator  to  be  used  in  connection  with  a  petroleum  lamp.  In  the 
absence  of  any  regulating  apparatus  an  incubating  oven  may  be  kept 
at  a  tolerably  uniform  temperature  by  personal  supervision — adjusting 
the  flame  of  the  lamp  and  its  distance  from  the  bottom  of  the  oven  ac- 
cording to  the  changes  in  the  external  temperature.  For  most  bac- 
teria a  variation  of  several  degrees  is  not  important,  so  long  as  the 
temperature  is  not  allowed  to  rise  above  37°  to  38°  C.  The  typhoid 
bacillus,  the  diphtheria  bacillus,  the  anthrax  bacillus,  the  pus  cocci, 
and  most  saprophytic  bacteria  grow  at  the  ordinary  room  temperature, 
and  may  therefore  be  cultivated  without  any  form  of  incubating  oven 
or  thermo-regulator. 


XL 
EXPERIMENTS  UPON  ANIMALS. 

THE  pathogenic  power  of  various  bacteria  has  been  demonstrated 
by  injecting  pure  cultures  into  susceptible  animals.  As  a  rule,  the 
herbivora  are  more  susceptible  than  the  carnivora,  and  this  is  per- 
haps to  be  explained  in  accordance  with  the  theory  of  natural  selec- 
tion. Carnivorous  animals  often  feed  upon  the  bodies  of  animals 
which  have  succumbed  to  infectious  diseases,  and  upon  dead  animals 
in  which  putrefactive  changes  have  commenced.  In  their  struggles 
with  each  other  they  are  wounded  by  teeth  and  claws  soiled  with  in- 
fectious material  which  would  cause  a  fatal  disease  if  inoculated  into 
the  more  susceptible  herbivorous  animals.  As  this  has  been  going 
on  for  ages,  we  may  suppose  that,  by  survival  of  the  fittest,  a  race 
tolerance  has  been  acquired.  The  lower  animals  have  their  own  in- 
fectious diseases,  some  of  which  are  peculiar  to  certain  species  and 
some  common  to  several.  Asa  rule,  the  specific  infectious  disea»  - 
of  man  cannot  be  transmitted  to  lower  animals,  and  man  is  not  sub- 
ject to  the  diseases  of  the  same  class  which  prevail  among  animals. 
But  certain  diseases  furnish  an  exception  to  this  general  rule.  Thus 
tuberculosis  is  common  to  man  and  several  of  the  lower  animals  : 
relapsing  fever  may  by  inoculation  be  transmitted  to  monkeys ; 
diphtheria  may  be  transmitted  to  pigeons  and  guinea-pigs.  On  the 
other  hand,  anthrax  and  glanders  may  be  contracted  by  man  as  a 
result  of  accidental  inoculation  or  contact  with  an  infected  animal. 

Nearly  allied  species  sometimes  present  very  remarkable  differ- 
ences as  to  susceptibility.  Thus  the  bacillus  of  mouse  septicaemia  is 
fatal  to  house  mice  but  not  to  field  mice,  while,  on  the  other  hand. 
field  mice  are  killed  by  the  bacillus  of  glanders  and  house  mice  are 
immune  from  this  pathogenic  bacillus. 

The  animals  most  commonly  used  for  testing  the  pathogenic 
po  \\-rr  <>f  bacteria  are  the  mouse,  the  guinea-pig,  and  the  rabbit. 
Domestic  fowls  and  pigeons  arc  also  useful  for  certain  experiments. 
The  dog  and  the  rat  are  of  comparatively  little  use  on  account  of 
their  slight  susceptibility. 


EXPERIMENTS   UPON  ANIMALS. 


95 


Inoculations  are  made  directly  into  the  circulation  through  a 
vein,  into  the  subcutaneous  connective  tissue,  or  into  one  of  the 
serous  cavities — usually  the  peritoneal. 

The  ordinary  hypodermic  syringe  may  be  used  in  making  injec- 
tions, but  this  is  difficult  to  sterilize  on  account  of  the  leather  piston, 
and  complications  are  liable  to  arise  from  its  use  which  it  is  best  to 
avoid.  The  best  way  to  sterilize  a  piston  syringe  is  to  wash  it  thor- 
oughly with  a  solution  of  bichloride  of  mercury  of  1  : 1,000,  and  then 
to  remove  every  trace  of  bichloride  by  washing  in  alcohol.  But  one 
never  feels  quite  sure  that  the  most  careful  washing  will  insure  steril- 
ization, and  it  is  best  to  use  a  syringe  which  may  be  sterilized  by 


FIG.  71. 


heat,  such  as  that  of  Koch,  shown  in  Fig.  71.  In  this  the  metal  point 
and  glass  tube  are  easily  sterilized  in  a  hot-air  oven.  Fluid  is  drawn 
into  the  syringe  and  forced  out  of  it  by  a  rubber  ball  which  has  a 
perforation  to  be  covered  by  the  finger. 

The  writer  has  for  some  years  been  in  the  habit  of  making  injec- 
tions in  animals  with  an  improvised  glass  syringe.  This  is  made 
from  a  piece  of  glass  tubing  in  the  same  form  as  the  collecting  tubes 
heretofore  described.  A  bulb  is  blown  at  one  end  of  the  tube,  and 
the  other  end  is  drawn  out  to  form  a  slender  tube  which  serves  as  the 


FIG.  72. 


needle  of  the  syringe  (Fig.  72).  By  gently  heating  the  bulb  in  an 
alcohol  lamp  and  immersing  the  open  end  of  the  capillary  tube  in 
the  fluid  to  be  injected,  this  rises  into  the  syringe  as  the  expanded  air 
cools.  Having  introduced  the  glass  point  beneath  the  skin  or  into 
the  cavity  of  the  abdomen  of  the  animal  to  be  injected,  the  contents 
of  the  tube  are  forced  out  by  again  heating  the  bulb  by  means  of  a 
small  alcohol  lamp.  The  glass  point  is  easily  forced  through  the 
thin  skin  of  a  mouse  or  of  a  young  rabbit ;  but  for  animals  with  a 
thicker  skin  it  is  necessary  to  cut  through,  or  nearly  through,  the 
skin  with  some  other  instrument.  A  small  pair  of  curved  scissors 
answers  very  well  for  this  purpose. 


96  EXPERIMENTS   UPON  ANIMALS. 

Generally,  in  making  injections  into  animals,  it  is  customary  to 
remove  the  hair  for  some  distance  around  the  point  of  inoculation 
with  scissors  and  razor,  and  then  to  sterilize  the  surface  by  careful 
washing  with  a  solution  of  bichloride  of  mercury.  This  precaution 
is  necessary  in  researches  in  which  pathogenic  bacteria  are  being 
tested,  in  order  to  remove  any  possibility  of  accidental  inoculation 
with  germs  other  than  those  under  investigation,  and,  as  a  conse- 
quence, a  mistaken  inference  as  to  the  pathogenic  action  of  the  spe- 
cies under  investigation.  But  when  we  know  the  specific  pathogenic 
power  of  a  certain  microorganism  it  is  hardly  necessary  to  take  this 
precaution,  as  a  few  drops  of  culture  will  contain  millions  of  the  bac- 
teria, while  contamination,  if  it  occurs  from  the  surface  of  the  body, 
must  be  by  a  comparatively  small  number  of  bacteria,  which  are 
likely  to  be  of  a  harmless  kind  which  will  have  no  influence  on  the 
result  of  the  experiment. 

Instead  of  sterilizing  the  surface,  the  writer  usually  clips  away  a 
small  portion  of  skin  with  curved  scissors,  not  cutting  deep  enough 
to  draw  blood,  but  leaving  a  bare  surface  through  which  the  point  of 
the  syringe  can  be  introduced  with  very  little  danger  of  carrying  Bac- 
teria into  the  connective  tissue  other  than  those  contained  in  the 
syringe. 

In  making  injections  into  the  peritoneal  cavity  care  must  be  taken 
not  to  wound  the  liver  or  the  distended  stomach.  The  intestine  is 
not  very  likely  to  be  wounded,  as  it  slips  out  of  the  way.  By  seizing 
a  longitudinal  fold  of  the  abdominal  wall  and  pushing  the  point  of 
the  syringe  quite  through  it,  and  then  releasing  the  fold  and  care- 
fully withdrawing  the  instrument  until  the  point  remains  in  the 
cavity,  the  danger  of  wounding  the  intestine  will  be  reduced  to  a 
minimum. 

Injections  into  the  circulation  are  made  by  exposing  a  vein  and 
carefully  introducing  the  needle  of  the  syringe  in  the  direction  of 
tlir  blood  current.  Care  must  of  course  be  taken  not  to  inject  air. 
In  the  rabbit  one  of  the  large  veins  of  the  ear  may  be  conveniently 
I" -net  rated  1,\  t lie  point  of  a  hypodermic  syringe  without  any  piv- 
N  i«  -us  dissection.  The  ear  is  first  washed  with  a  solution  of  bichloride 
of  mercury  or  simply  with  warm  water.  The  animal  had  better  be 
<H'  fully  wrapped  in  a  towel  to  control  its  movements.  The  veins 
an-  distended  by  compressing  them  near  the  base  of  the  ear.  When 
the  point  of  the  needle  has  not  been  properly  introduced,  and  the 
tin  id  to  be  injected  escapes  in  the  surrounding  connective  tissue,  it 
will  commonly  be  best  to  withdraw  the  syringe  and  make  the 
attempt  upon  another  vein.  As  pointed  out  by  Abbott,  the  needle 
of  the  syringe  should  be  ground  flat  at  the  point,  and  not  curved  as 
is  commonly  the  case. 


EXPERIMENTS   UPON   ANIMALS.  97 

Large  quantities  of  fluid  may  be  injected  into  the  cavity  of  the 
abdomen  or  into  the  circulation  by  slowly  forcing  the  fluid  through 
a  slender  canula,  properly  introduced,  which  is  coupled  with  a  large 
syringe  by  means  of  rubber  tubing,  or  with  a  glass  receptacle  from 
which  the  fluid  is  forced  by  the  pressure  of  air  pumped  in  with  a 
rubber  hand  ball. 

Mice  are  usually  injected  subcutaneously  near  the  tail.  The 
little  animal  is  first  seized  by  a  long  pair  of  forceps,  or  "mouse 
tongs,"  and  the  hair  is  clipped  away  on  the  back  just  above  the  tail. 
If  solid  material  is  to  be  introduced  a  little  pocket  is  made  with  scis- 
sors or  with  a  lancet,  into  which  the  infectious  material  is  carried  by 
means  of  a  platinum  needle  or  slender  forceps.  Liquids  may  be  in- 
jected by  the  little  glass  syringe  heretofore  described,  the  point  of 
which  is  easily  forced  through  the  skin. 

Pasteur's  method  of  inoculating  rabbits  with  the  virus  of  hydro- 
phobia consists  in  trephining  the  skull  and  injecting  the  material 
beneath  the  dura  mater.  An  incision  through  the  skin  is  first  made 
to  one  side  of  the  median  line  a  short  distance  back  of  the  eyes. 
The  edges  of  the  wound  are  separated,  and  a  small  trephine  (five  or 
six  millimetres  in  diameter)  is  used  to  remove  a  button  of  bone.  The 
emulsion  of  spinal  cord  from  a  hydrophobic  animal  is  then  carefully 
injected  beneath  the  dura  mater — two  or  three  drops  will  be  sufficient. 
The  wound  is  washed  out  with  a  two-per-cent  solution  of  carbolic 
acid  and  closed  with  a  couple  of  sutures. 

Injections  into  the  intestine  are  made  by  carefully  opening  the 
abdomen  with  antiseptic  precautions,  gently  seizing  a  loop  of  the  in- 
testine, and  passing  the  point  of  the  syringe  through  its  walls  ;  the 
loop  is  then  returned  and  the  incision  in  the  walls  of  the  abdomen 
carefully  closed  with  sutures  and  dressed  antiseptically. 

Inoculations  into  the  anterior  chamber  of  the  eye  of  rabbits  and 
other  animals  have  frequently  been  practised,  and  offer  certain  ad- 
vantages in  the  study  of  the  local  effects  of  pathogenic  microorgan- 
isms. The  animal  should  be  fastened  to  an  operating  board,  belly 
down,  and  its  head  held  by  an  assistant,  who  at  the  same  time  holds 
the  eyelids  apart.  The  conjunctiva  is  seized  with  forceps  to  steady 
the  eye,  and  an  incision  about  two  millimetres  long  is  made  through 
the  cornea  with  a  cataract  knife.  Through  this  opening  a  small 
quantity  of  a  liquid  culture  may  be  injected,  or  a  bit  of  solid  material 
introduced  with  slender  curved  forceps. 

Ordinary  injections  give  but  little  pain  and  do  not  call  for  the  use 
of  an  anaesthetic.  When  anaesthesia  is  required  ether  will  usually 
be  preferable  to  chloroform.  Rabbits,  especially,  are  very  apt  to  die 
from  chloroform,  no  matter  how  carefully  it  may  be  administered. 
Dogs,  rats,  and  mice  stand  ether  very  well.  The  smaller  animals 
7 


98  EXPERIMENTS   UPON  ANIMALS. 

may  be  brought  under  the  anaesthetic  by  placing  them  in  a  covered 
jar  into  which  a  pledget  of  cotton  wet  with  ether  has  been  dropped. 
Before  making  injections  into  the  anterior  chamber  of  the  eye  it  is 
well  to  use  a  two-per-cent  solution  of  cocaine  as  a  local  anaesthetic. 

Mice  which  have  been  inoculated  are  usually  kept  in  a  glass  jar 
having  a  wire-gauze  cover.  A  quantity  of  cotton  is  put  into  the  jar 
to  serve  as  a  shelter  for  the  little  animal,  and  it  is  well  to  partly  fill 
the  jar  with  dry  sawdust.  Larger  animals  are  kept  in  suitable  cages 
of  wire  or  wood,  and,  as  a  rule,  each  one  should  be  kept  in  a  separate 
cage  while  under  observation  after  an  inoculation  experiment. 

In  experimenting  upon  animals  the  following  points  should  be 
kept  in  view  and  noted  : 

(a)  The  age  and  weight  of  the  animal.     Young  animals  are,  as 
a  rule,  more  susceptible  than  older  ones,  and  with  many  pathogenic 
bacteria  the  lethal  dose  of  a  culture  bears  some  relation  to  the  size 
of  the  animal. 

(b)  The  point  of  inoculation.     Injections  into  the  circulation 
are  generally  more  promptly  fatal  and  require  a  smaller  dose  than 
those  into  a  serous  cavity  or  into  the  connective  tissue.     Pathogenic 
bacteria  introduced  into  the  abdominal  cavity  reach .  the  circulation 
more  promptly  than  those  injected  subcutaneously.     But  certain 
microorganisms  owe  their  pathogenic  power  to  the  local  effect  about 
the  point  of  inoculation  and  the  absorption  of  toxic  products  formed 
in  the  limited  area  invaded,  and  do  not  enter  the  general  circulation, 
or  at  least  do  nqt  multiply  in  the  circulating  fluid,  and  quickly  dis- 
appear from  it. 

(b)  The  age  of  the  culture  injected.  Old  cultures  sometimes 
have  greater  and  sometimes  less  pathogenic  potency  than  recent  cul- 
tures. Some  kinds  of  virus  become  "  attenuated  "  when  kept.  But 
when  the  pathogenic  power  depends  chiefly  upon  toxic  products 
formed  during  the  growth  of  the  bacteria,  old  cultures  are,  as  a  rule, 
more  potent  than  those  recently  made. 

(d)  The  medium  in  which  the  pathogenic  bacteria  are  .s/f.v- 
pended.     Cultures  in  albuminous  media,  like  blood  serum,  are  in 
some  cases  more  potent  than  bouillon  cultures  ;  and  the  virulence  of 
several  pathogenic  bacteria  is  greatly  intensified  by  successive  cul- 
tures— by  inoculation — in  the  bodies  of  susceptible  animals.     Ogston 
found  that  pus  cocci  cultivated  in  the  interior  of  eggs  had  an  in- 
creased virulence.     According  to  Arloing,  Cornevin,  and  Thomas, 
the  activity  of  a  culture  of  the  bacillus  of  symptomatic  anthrax  is 
doubled  by  adding  one-five-hundredth  part  of  lactic  acid  to  the  cul- 
ture fluid. 

(e)  The  quantity  injected  is  evidently  an  essential  point  when 
the  result  depends  largely  upon  the  toxic  products  formed  in  the  cul- 


EXPERIMENTS   UPON   ANIMALS.  99 

ture  medium.  It  is  also  an  essential  point  when  pathogenic  bacteria 
are  injected  which  kill  susceptible  animals  in  very  minute  doses,  for 
it  has  been  shown  by  the  experiments  of  Watson  Cheyne  and  others 
that  in  the  case  of  some  of  these,  at  least,  there  is  a  limit  below 
which  infection  does  not  occur. 

Inoculated  animals  should  be  carefully  observed,  and  a  note 
made  of  every  symptom  indicating  a  departure  from  the  usual  con- 
dition of  health,  such  as  fever,  less  of  activity,  loss  of  appetite, 
weakness,  emaciation,  diarrhoea,  convulsions,  dilated  pupils,  the  for- 
mation of  an  abscess  or  a  diffuse  cellulitis  extending  from  the  point 
of  inoculation,  etc.  The  temperature  is  usually  taken  in  the  rectum. 
The  temperature  of  small  animals,  like  rabbits  and  guinea-pigs,  va- 
ries considerably  as  a  result  of  external  conditions.  In  the  rabbit 
the  normal  temperature  may  be  given  as  about  102°  to  103°  F,  ;  in 
the  guinea-pig  it  is  a  little  lower. 

In  making  a  post-mortem  examination  of  an  inoculated  animal  it 
is  best  to  stretch  it  out  on  a  board,  belly  up,  by  tying  its  legs  to  nails 
or  screws  fastened  in  the  margin  of  the  board.  When  the  abdomen 
is  dirty,  as  is  usually  the  case,  it  should  be  carefully  washed  with  a 
disinfecting  solution.  An  incision  through  the  skin  is  then  made  in 
the  median  line  the  full  length  of  the  body,  and  the  skin  is  dis- 
sected back  so  as  to  expose  the  anterior  walls  of  the  abdomen  and 
thorax.  These  cavities  are  then  carefully  opened  with  a  sterilized 
knife  or  scissors,  and  the  various  organs  and  viscera  examined.  At- 
tention should  also  be  given  to  the  appearances  at  the  point  of  in- 
oculation. To  ascertain  whether  the  microorganism  injected  has 
invaded  the  blood,  smear  preparations  should  be  made  with  blood 
obtained  from  a  vein  or  from  one  of  the  cavities  of  the  heart.  It 
will  be  well  also  to  make  a  smear  preparation  from  a  cut  surface  of 
the  liver  and  spleen.  In  the  various  forms  of  acute  septicaemia  the 
spleen  is  usually  found  to  be  enlarged.  If  but  few  microorganisms 
are  present  in  the  blood  and  tissues  they  may  escape  observation  in 
stained  smear  preparations,  and  it  will  be  necessary  to  make  cultures 
to  demonstrate  their  presence.  A  little  blood  from  a  vein  or  from 
one  of  the  cavities  of  the  heart  is  transferred,  by  means  of  a  plati- 
num loop  (ose)  or  a  sterilized  collecting  tube  (see  page  38),  to  a 
test  tube  containing  liquefied  nutrient  gelatin  or  agar-agar,  and  an 
Esmarch  roll  tube  is  made.  This  is  put  aside  for  the  development  of 
colonies  from  any  scattered  bacteria  which  may  be  present.  As  a 
rule,  it  will  be  best  to  make  agar  cultures,  as  these  can  be  placed  in 
the  incubating  oven  at  35°  to  38°  C.  Stick^cultures  may  also  be 
made  and  will  serve  to  show  the  presence  of  microorganisms,  but 
will  not  give  information  as  to  how  numerous  they  may  be.  The 
roll  tube  also  has  the  advantage  of  showing  whether  there  is  a 


100  EXPERIMENTS   UPON   ANIMALS. 

mixed  infection  or  whether  a  pure  culture  of  a  single  microorganism 
is  obtained  from  the  blood.  In  the  same  way  cultures  may  be  made 
from  material  obtained  from  the  liver  or  spleen,  and  it  may  happen 
that  one  or  both  of  these  organs  contain  bacteria  when  none  are 
found  in  the  blood.  Before  passing  the  platinum  needle  or  collect- 
ing tube  into  the  organ,  the  surface,  which  has  been  more  or  less  ex- 
posed to  contamination,  should  be  sterilized  by  applying  to  it  a  hot 
spatula ;  then  at  the  moment  of  lifting  the  spatula  the  sterilized 
needle  is  introduced  into  the  interior  of  the  organ,  and  the  blood  and 
crushed  tissue  adhering  to  it  at  once  carried  over  to  the  culture  me- 
dium. Or  blood  obtained  with  proper  precautions  from  a  vein,  a 
cavity  of  the  heart,  or  the  interior  of  the  spleen  or  liver,  may  be 
used  to  inoculate  another  animal. 

Animals  are  also  sometimes  inoculated  by  excoriating  the  cutis 
as  in  vaccination.  They  may  also,  in  rare  cases,  be  infected  by  in- 
troducing cultures  into  the  stomach,  either  mixed  with  the  food  in- 
gested or  by  injection  through  a  tube.  Infection  by  inhalation  is 
accomplished  by  causing  the  animal  to  respire  an  atmosphere,  in  a 
properly  enclosed  space,  in  which  the  pathogenic  organism  is  sus- 
pended, by  the  use  of  a  spray  apparatus  for  liquid  cultures,  or 
some  form  of  powder  blower  for  powders  containing  the  bacteria  in 
a  desiccated  condition. 

One  method  of  obtaining  a  pure  culture  of  pathogenic  bacteria 
consists  in  the  inoculation  of  susceptible  animals  with  material  con- 
taining a  pathogenic  species  in  association  with  others  which  are  not. 
When  the  blood  is  invaded  by  the  pathogenic  species  and  the  animal 
dies  from  an  acute  septicaemia,  we  may  usually  obtain  a  pure  cul- 
ture by  inoculating  a  suitable  culture  medium  with  a  minute  drop  of 
blood  taken  from  a  vein  or  from  one  of  the  cavities  of  the  heart. 
Sometimes,  however,  a  mixed  infection  occurs  and  some  other  mi- 
croorganism is  associated  in  the  blood  with  that  one  which  was  tlir 
immediate  cause  of  the  death  of  the  animal. 


XII. 
PHOTOGRAPHING  BACTERIA. 

WELL-MADE  photomicrographs  are  unquestionably  superior  to 
drawings  made  by  hand  as  a  permanent  record  of  morphological 
characters.  This  being  the  case,  bacteriologists  would  no  doubt  re- 
sort to  this  method  more  generally  but  for  the  technical  difficulties 
and  the  time  and  patience  required  in  overcoming  these.  Koch,  in 
his  earlier  studies,  gave  much  time  to  photographing  bacteria,  and 
with  very  remarkable  success.  In  his  work  on  "Traumatic  Infec- 
tive Diseases  "  (1878)  he  says  : 

"With  respect  to  the  illustrations  accompanying  this  work,  I 
must  here  make  a  remark.  In  a  former  paper  *  on  the  examination 
and  photographing  of  bacteria  I  expressed  the  wish  that  observers 
would  photograph  pathogenic  bacteria  in  order  that  their  representa- 
tions of  them  might  be  as  true  to  nature  as  possible.  I  thus  felt 
bound  to  photograph  the  bacteria  discovered  in  the  animal  tissues  in 
traumatic  infective  diseases,  and  I  have  not  spared  trouble  in  the 
attempt.  The  smallest,  and  in  fact  the  most  interesting  bacteria, 
however,  can  only  be  made  visible  in  animal  tissues  by  staining 
them  and  by  thus  gaining  the  advantage  of  color.  But  in  this  case 
the  photographer  has  to  deal  with  the  same  difficulties  as  are  expe- 
rienced in  photographing  colored  objects — e.g.,  colored  tapestry. 
These  have,  as  is  well  known,  been  overcome  by  the  use  of  colored 
collodion.  This  led  me  to  use  the  same  method  for  photographing 
stained  bacteria,  and  I  have,  in  fact,  succeeded,  by  the  use  of  eosin- 
collodion,  and  by  shutting  off  portions  of  the  spectrum  by  colored 
glasses,  in  obtaining  photographs  of  bacteria  which  had  been  stained 
with  blue  and  red  aniline  dyes.  Nevertheless,  from  the  long  ex- 
posure required  and  the  unavoidable  vibrations  of  the  apparatus,  the 
picture  does  not  have  sharpness  of  outline  sufficient  to  enable  it  to  be 
of  use  as  a  substitute  for  a  drawing,  or,  indeed,  even  as  evidence  of 
what  one  sees.  For  the  present,  therefore,  I  must  abstain  from  pub- 
lishing photographic  representations ;  but  I  hope,  at  a  subsequent 
period  when  improved  methods  allow  a  shorter  exposure,  to  be  able 
to  remedy  this  defect." 

1  The  paper  referred  to  is  published  in  Cohn's  "Beitrage  zur  Biologic  d.  Pflanzen." 


102  PHOTOGRAPHING   BACTERIA. 

Since  the  above  was  written  considerable  progress  has  been  made 
in  removing  the  technical  difficulties,  and  a  few  bacteriologists  have 
succeeded  in  making  very  satisfactory  photomicrographs.  As  speci- 
mens of  what  may  be  done  with  the  best  apparatus  and  the  highest 
degree  of  skill,  we  may  call  attention  to  the  pkotomicrographs  in 
the  Atlas  der  Bakterienkunde  of  Frankel  and  Pfeiffer,  and  those 
of  Roux  in  the  Annales  of  the  Pasteur  Institute.  The  writer,  also, 
has  devoted  much  time  to  making  photomicrographs  which  have 
served  as  illustrations  for  several  of  his  published  works. 

Those  who  have  had  no  practical  experience  in  making  photo- 
micrographs are  apt  to  expect  too  much  and  to  underestimate  the 
technical  difficulties.  Objects  which  under  the  microscope  give  a 
beautiful  picture,  which  we  desire  to  reproduce  by  photography,  may 
be  entirely  unsuited  for  the  purpose.  In  photographing  with  high 
powers  it  is  necessary  that  the  objects  to  be  photographed  be  in  a 
single  plane  and  not  crowded  together  or  overlying  each  other. 
For  this  reason  photographing  bacteria  in  sections  presents  special 
difficulties,  and  satisfactory  results  can  only  be  obtained  when  the 
sections  are  extremely  thin  and  the  bacteria  well  stained.  Even 
with  the  best  preparations  of  this  kind  much  care  must  be  taken  in 
selecting  a  field  for  photography.  It  must  be  remembered  that  the 
expert  microscopist,  in  examining  a  section  with  high  powers,  has 
his  finger  on  the  fine  adjustment  screw  and  focuses  up  and  down  to 
bring  different  planes  into  view.  He  is  in  the  habit  of  fixing  his  at- 
tention on  that  part  of  the  field  which  is  in  the  focus  and  disregard- 
ing the  rest.  But  in  a  photograph  the  part  of  the  field  not  in  focus 
appears  in  a  prominent  way  which  mars  the  beauty  of  the  picture. 
In  a  cover-glass  preparation  made  from  a  pure  culture,  when  tin* 
bacteria  are  well  distributed,  this  difficulty  does  not  present  itself,  as 
the  bacteria  are  all  lying  in  a  single  plane;  but  the  portion  of  the  field 
which  can  be  shown  at  one  time  is  limited  by  the  spherical  aberra- 
tion of  the  objective,  which  the  makers  do  not  seem  able  to  overcome 
in  high-power  lenaee  of  wide  angle,  at  least  not  without  loss  of  de- 
lining  power. 

Usually  preparations  of  bacteria  are  stained  for  photography, 
but  with  some  of  the  larger  forms,  such  as  the  anthrax  bacillus, 
very  satisfactory  photomicrographs  may  be  made  from  unstained 
preparations.  In  this  case  a  small  quantity  of  a  recent  culture  is 
put  upon  a  slide,  covered  with  a  thin  cover  glass,  and  placed  at  once 
1 1  p o 1 1  t  he  stage  of  the  microscope.  The  main  difficulty  to  be  encoun- 
tered results  from  the  change  of  location  of  the  suspended  bacteria 
resulting  from  the  pressure  of  the  objective  in  focussing.  Motile 
lueteria,  of  course,  cannot  be  photographed  in  this  way  without  first 
arresting  their  movements  by  means  of  some  germicidai  agent; 


PHOTOGRAPHING  BACTERIA.  103 

and  in  general  it  will  be  found  more  satisfactory  to  fix  the  micro- 
organisms to  be  photographed  to  a  slide  or  cover  glass  by  desiccation 
and  heat,  and  to  stain  them  with  one  of  the  aniline  colors. 

Objects  which  are  opaque  cannot  be  photographed  by  transmitted 
light,  and  objects  which  have  a  deep  orange  or  red  color  are  practi- 
cally opaque  for  the  actinic  rays  which  are  at  the  violet  end  of  the 
spectrum.  Such  objects  simply  intercept  the  light,  but  this  gives 
the  outlines,  and,  where  there  are  no  details  of  structure,  is  all  that 
is  required  to  illustrate  the  form  and  mode  of  grouping.  Softer  and 
more  satisfactory  photomicrographs  of  bacteria  are  made  when  the 
staining  is  not  such  as  to  entirely  arrest  the  actinic  rays.  Among 
the  aniline  colors  Bismarck  brown  and  vesuvin  are  the  most  suitable, 
care  being  taken,  with  the  larger  bacteria  especially,  not  to  make 
the  staining  too  intense.  Objects  which  are  transparent  for  the  ac- 
tinic rays,  or  nearly  so,  give  a  very  feeble  photographic  image,  or 
none  at  all,  on  account  of  the  want  of  contrast  in  the  impression 
made  upon  the  sensitive  plate.  This  is  the  case  when  we  attempt  to 
photograph,  by  ordinary  white  light,  objects  which  are  stained  violet 
or  blue.  But  this  want  of  contrast  in  the  negative  can  be  overcome 
by  the  use  of  specially  prepared  plates  and  colored  screens  of  glass 
interposed  between  the  object  and  the  source  of  light.  The  so-called 
orthochromatic  plates  are  more  sensitive  to  the  rays  toward  the  red 
end  of  the  spectrum  than  ordinary  plates.  They  are  prepared  by 
treating  the  plates  with  a  solution  of  eosin,  of  erythrosin,  or  of  rose 
bengal  (Vogel),  and  may  now  be  purchased  in  this  country  from 
dealers  in  dry  plates.  If  we  shut  off  the  violet  rays  by  the  use  of  a 
yellow  screen,  objects  having  a  yellow  or  orange  color  may  be  pho- 
tographed upon  orthochromatic  plates,  although  the  time  of  exposure 
will  be  quite  long  owing  to  the  comparatively  feeble  actinic  power 
of  the  yellow  rays. 

We  may  also  make  photomicrographs  of  objects  stained  with 
methylene  blue  or  with  fuchsin,  because  objects  stained  with  these 
colors  are  opaque  for  the  rays  from  the  red  end  of  the  spectrum,  and 
sufficiently  so  with  yellow  light  to  give  a  good  photographic  con- 
trast. Frankel  and  Pfeiffer  recommend  the  use  of  a  green  light-fil- 
ter (green  glass  screen)  for  all  preparations  stained  with  methyl  vio- 
let, fuchsin,  or  methylene  blue;  and  for  brown-stained  preparations  a 
pure  blue  light.  The  writer  has  been  in  the  habit  of  using  a  yellow 
glass  screen  for  fuchsin-stained  preparations,  and  has  had  excellent 
results,  but  the  time  of  exposure  is  necessarily  long.  A  yellow  glass 
screen  may  be  prepared  by  dissolving  tropaeolin  in  negative  varnish, 
and  pouring  this  upon  a  clean  glass  slide,  where  it  is  permitted  to 
<lry. 

To  show  bacteria  in  photographs  in  a  satisfactory  manner  we 


104  PHOTOGRAPHING   BACTERIA. 

require  an  amplification  of  five  hundred  to  one  thousand  diameters  : 
and  as  it  is  often  desirable  to  make  comparisons  as  to  the  dimen- 
sions of  microorganisms  which  resemble  each  other  in  form,  it  is 
best  to  adopt  a  standard  amplification.  The  writer  has  himsel: 
adopted,  and  would  recommend  to  others,  a  standard  amplification 
of  one  thousand  diameters.  This  is  about  as  high  a  magnifying 
power  as  we  can  get  with  satisfactory  definition,  or  as  we  require, 
and  it  is  a  convenient  number  when  measurements  are  made  from 
the  photograph.  The  beginner,  after  having  put  his  apparatus  in 
position,  should  focus  the  lines  of  a  stage  micrometer  upon  the 
screen  with  the  optical  apparatus  which  he  proposes  to  use  ;  then  by 
moving  the  screen  forward  or  back  as  required,  and  carefully  focus- 
sing the  lines,  he  will  ascertain  what  is  the  position  of  the  screen  for 
exactly  one  thousand  diameters.  If  the  stage  micrometer  is  ruled 
with  lines  which  are  one  one-thousandth  of  an  inch  apart,  it  is  evi- 
dent that  when  projected  upon  the  screen  they  should  be  one  inch 
apart  to  make  the  amplification  one  thousand  diameters.  But  it 
must  be  remembered  that  any  change  in  the  position  of  the  optical 
combination  will  change  the  amplification.  If,  therefore,  the  cover 
correction  of  the  objective  is  changed,  or  the  position  of  the  eyepiece 
—if  one  is  used — it  will  be  necessary  to  again  adjust  the  distance  of 
the  screen. 

Apparatus  required. — A  first-class  immersion  objective  of  one- 
twelfth  of  an  inch  or  higher  power,  a  substantial  stand  which  can  bo 
placed  in  a  horizontal  position,  and  a  camera  which  can  be  coupled 
with  the  microscope  tube,  are  the  essential  pieces  of  apparatus.  If 
sunlight  is  to  be  used  a  heliostat  will  also  be  required. 

The  oil-immersion  objectives  of  any  good  maker  may  be  used, 
but  the  apochromatic  objectives  and  projection  eyepieces  of  Carl 
Zeiss,  of  Jena,  are  especially  to  be  recommended.  Indeed,  those  who 
can  afford  it  will  do  well  to  get  Zeiss'  complete  apparatus,  which 
includes  a  stand  having  a  mechanical  stage  and  a  camera  mounted 
upon  a  metal  frame  conveniently  provided  with  focussing  appliances, 
etc.  However,  good  work  may  be  done  with  less  expensive  appa- 
ratus. 

The  stand  should  be  substantial  and  well  made,  with  a  delicate, 
fine  adjustment.  A  mechanical  stage  is  not  essential,  but  is  a  great 
convenience  in  finding  and  adjusting  to  the  centre  of  the  screen  a 
satisfactory  field  to  photograph.  The  substage  should  be  provided 
with  a  good  apochromatic  condenser,  and  with  appliances  for  moving 
the  condensing  lens  forward  and  back  and  for  centring  it,  with  dia- 
phragms, etc. 

By  the  use  of  a  high-power  objective,  like  the  one-eighteenth-inc-h 
oil-immersion  of  Zeiss,  the  desired  amplification  may  be  obtained 


PHOTOGRAPHING   BACTERIA.  105 

without  the  use  of  an  eyepiece  ;  and,  as  a  rule,  it  is  best  not  to  use 
an  ordinary  eyepiece  to  secure  increased  amplification,  as  this  is  ob- 
tained at  the  expense  of  definition.  But  an  amplifier  may  be  used  in 
the  tube  of  the  microscope,  as  first  recommended  by  Woodward.  In 
this  case  the  amplifier  must  be  carefully  adjusted  with  reference  to 
the  distance  of  the  screen,  to  secure  the  best  possible  definition. 

The  projection  eyepieces  of  Zeiss  are  constructed  especially  for 
photography  and  possess  a  decided  advantage.  By  the  use  of  his 
three-millimetre  apochromatic  oil-immersion  objective  and  projec- 
tion eyepiece  No.  3  we  may  obtain  an  amplification  of  one  thousand 
diameters  with  excellent  definition. 

Light. — Sunlight  is  in  many  respects  the  most  satisfactory  for 
photography,  but  has  the  disadvantage  that  it  is  not  always  available. 
In  some  sections  of  the  country  weeks  may  pass  without  a  single 
clear  day  suitable  for  making  photomicrographs.  In  addition  to  the 
uncertainty  arising  from  cloudy  weather,  we  have  to  contend  with 
the  fact  that  the  sun  is  only  available  for  use  with  a  heliostat  for  a 
limited  time  during  each  day,  and  that  this  time  is  greatly  restricted 
in  Northern  latitudes  during  the  winter  months.  When  sunlight  is 
to  be  employed  the  microscope  and  camera  must  be  set  up  in  a  room 
having  a  southern  exposure  on  a  line  corresponding  with  the  true 
meridian  of  the  place.  The  heliostat  is  placed  outside  the  window  in 
such  a  position  that  when  properly  adjusted  the  light  of  the  sun  will 
fall  upon  the  condenser  attached  to  the  substage  of  the  microscope. 
The  condenser  must  be  carefully  centred,  so  that  the  circle  of  light 
falling  upon  the  screen  shall  be  uniform  in  intensity  and  outline. 

The  calcium,  magnesium,  or  electric  light  may  be  used  as  a  sub- 
stitute for  sunlight,  but  they  are  all  rather  expensive,  unless,  in  the 
case  of  the  electric  light,  a  suitable  current  is  available  without  the 
expense  of  generating  it  for  the  special  purpose  in  view.  The  writer 
has  obtained  very  good  results  with  the  calcium  light,  but  has  no  ex- 
perience in  the  use  of  the  electric  light.  Woodward,  as  a  result  of 
extended  experiments,  arrived  at  the  conclusion  that  "  the  electric 
light  is  by  far  the  best  of  all  artificial  lights  for  the  production  of 
photomicrographs. "  He  used  a  Grove  battery  of  fifty  elements  to 
generate  the  current,  and  a  Duboscq  lamp.  The  current  from  a 
dynamo  would  no  doubt  be  much  cheaper  and  more  conveniently 
used,  if  an  electric-lighting  plant  was  in  the  vicinity. 

The  apparatus  shown  in  Fig.  73  was  designed  by  Mr.  Pringle  for 
the  use  of  the  calcium  light.  It  will  serve  to  illustrate  the  arrange- 
ment of  the  microscope  and  camera  in  connection  with  any  other 
light  as  well.  An  oil  lamp  may  be  placed  in  the  position  of  the  oxy- 
hydrogen  burner  ;  or,  if  sunlight  is  to  be  employed,  a  heliostat  will 
be  placed  in  the  same  position. 


106 


PHOTOGRAPHING   BACTERIA. 


When  a  colored  screen  is  used  this  may  be  placed  either  before 
or  behind  the  condensing  lens — we  prefer  to  place  it  behind,  although 


Neuhauss  has  shown  that  it  makes  no  difference  in  the  length  of  th<« 
exposure. 

\\  6  <Miin.)t  iii  tin-  pivsimt  volume  iciv»>  full  d.'tails  with    ivtVivmv 


PHOTOGRAPHING  BACTERIA.  107 

to  the  technique  of  making  photomicrographs,  but  append  an  account 
of  a  form  of  apparatus  which  we  have  used  with  great  satisfaction  : 

"Photomicrography  by  Gaslight. — Those  who  have  had  much  experience 
in  making-  photomicrographs  will  agree  with  me  that  one  of  the  most  essen- 
tial elements  of  success  is  the  use  of  a  suitable  source  of  illumination. 

"  Without  question  the  direct  light  of  the  sun,  reflected  in  a  right  line  by 
the  mirror  of  a  heliostat,  is  the  most  economical  and,  in  some  respects,  the 
most  satisfactory  light  that  can  be  used.  But  we  cannot  command  this  light 
at  all  times  and  places,  and  it  often  happens  that,  when  we  are  ready  to  de- 
vote a  day  to  making  photomicrographs,  the  sun  is  obscured  by  clouds  or 
the  atmosphere  is  hazy.  Indeed,  in  some  latitudes  and  at  certain  seasons  of 
the  year  a  suitable  day  for  the  purpose  is  extremely  rare.  The  use  of  sun- 
light also  requires  a  room  having  a  southern  exposure  and  elevated  above  all 
surrounding  buildings  or  other  objects  by  which  the  direct  rays  of  the  sun 
would  be  intercepted.  For  these  reasons  a  satisfactory  artificial  light  is  ex- 
tremely desirable. 

"  The  oxyhydrogeii  lime  light,  the  magnesium  light,  and  the  electric  arc 
li^ht  have  all  been  employed  as  a  substitute  for  the  light  of  the  sun,  and  all 
give  satisfactory  results.  I  have  myself  made  rather  extensive  use  of  the 
'lime  light,' and  think  it  the  best  substitute  for  solar  light  with  which  I 
am  familiar.  But  to  use  it  continuously,  day  after  day,  is  attended  with 
considerable  expense,  and  the  frequent  renewal  of  the  supply  of  gas  which 
it  calls  for  is  an  inconvenience  which  one  would  gladly  dispense  with. 

"These  considerations  have  led  some  microscopists  to  use  an  oil  lamp  as 
the  source  of  illumination,  and  very  satisfactory  photomicrographs  with 
comparatively  high  power  have  been  made  with  this  cheap  and  convenient 
light.  But  in  my  experience  the  best  illumination  which  I  have  been  able 
to  secure  with  an  oil  lamp  has  called  for  very  long  exposures  when  working 
with  high  powers,  and,  as  most  of  my  photomicrographs,  of  bacteria  are 
made  with  an  amplification  of  one  thousand  diameters,  I  require  a  more 
powerful  illumination  than  I  have  been  able  to  secure  in  this  way.  And 
especially  so  because  of  the  fact  that  a  colored  screen  must  be  interposed, 
which  shuts  off  a  large  portion  of  the  actinic  rays,  on  account  of  the  staining 
agent  usually  employed  in  making  my  mounts.  The  most  satisfactory 
staining  agents  for  the  bacteria  are  an  aqueous  solution  of  fuchsin,  or  of 
methylene  blue,  or  of  gentian  violet;  and  all  of  these  colors  are  so  nearly 
transparent  for  the  actinic  rays  at  the  violet  end  of  the  spectrum  that  a 
satisfactory  photographic  contrast  cannot  be  obtained  unless  we  shut  off 
these  rays  by  a  colored  screen. 

"  I  am  in  the  habit  of  using  a  yellow  screen  for  my  preparations  stained 
with  f  uohsin  or  methylene  blue,  and  have  obtained  very  satisfactory  results 
with  the  orthochromatic  plates  manufactured  by  Carbutt,  of  Philadelphia, 
and  a  glass  screen  coated  with  a  solution  of  .tropaeolin  dissolved  in  gelatin. 

"  But  with  such  a  screen,  which  shuts  off  a  large  portion  of  the  actinic 
light  and  increases  the  time  of  exposure  three-  or  fourfold,  the  use  of  an 
oil  lamp  becomes  impracticable  with  high  powers,  on  account  of  the  feeble- 
ness of  the  illumination. 

"These  considerations  have  led  me  to  experiment  with  gaslight,  and  the 
simple  form  of  apparatus  which  I  am  about  to  describe  is  the  result  of  these 
experiments.  I  have  now  had  the  apparatus  in  use  for  several  months, 
during  which  time  I  have  made  a  large  number  of  very  satisfactory  photo- 
micrographs of  bacteria  from  f uchsm-stained  preparations  with  an  amplifica- 
tion of  one  thousand  diameters.  My  photographs  have  been  made  with  the 
three-millimetre  oil-immersion  apochromatic  objective  of  Zeiss  and  his  pro- 
jection eyepiece  No.  3.  I  use  a  large  Powell  and  Lealand  stand,  upon  the 
substage  of  which  I  have  fitted  an  Abbe' con  denser.  The  arrangement  of 
the  apparatus  will  be  readily  understood  by  reference  to  the  accompanying 
figure. 

"A  is  the  camera,  which  has  a  pyramidal  bellows  front  supported  by  the 


108 


PHOTOGRAPHING   BACTERIA. 


heavy  block  of  wood  B;  this  can  be  pushed  back  upon  the  baseboard  which 
supports  it,  so  as  to  allow  the  operator  to  place  his  eye  at  the  eyepiece  of  the 
microscope.  When  it  is  brought  forward  an  aperture  of  the  proper  size  ad- 
mits the  outer  extremity  of  the  eyepiece  and  shuts  off  all  light  except  that 
coming  through  the  objective.  C  is  the  microscope,  and  D  the  Abbe  con- 
denser, supported  upon  the  substage.  E  is  a  thick  asbestos  screen  for  pro- 
tecting the  microscope  from  the  heat  given  off  by  the  battery  of  gas  burners 
F.  This  asbestos  screen  has  an  aperture  of  proper  dimensions  to  admit  the 
light  to  the  condenser  D.  The  gas  burners  are  arranged  in  a  series,  with 
the  flat  portion  of  the  flame  facing  the  aperture  in  trie  asbestos  screen  E. 
The  concave  metallic  mirror  G  is  properly  placed  to  reflect  the  light  in  the 
desired  direction.  I  have  not  found  any  advantage  in  the  use  of  a  condens- 
ing lens  other  than  the  Abbe  condenser  upon  the  substage  of  the  microscope. 
The  focussing  is  accomplished  by  means  of  the  rod  I,  which  carries  at  one 
extremity  a  grooved  wheel,  H,  which  is  connected  with  the  fine  adjustment 
screw  of  the  microscope  by  means  of  a  cord. 

"  The  focussing  wheel  J  may  be  slipped  along  the  rod  I  to  any  desired 
position,  and  is  retained  in  place  by  a  set  screw.     The   rod  I  is  supported 


Fio.  74. 

above  the  camera  oy  arms  depending  from  the  ceiling,  or  by  upright  arms 
attached  to  the  baseboard. 

44 1  have  lost  many  plates  from  a  derangement  of  the  focal  adjustment 
resulting  from  vibrations  caused  by  the  passing  of  loaded  wagons  in  the 
street  adjoining  the  laboratory  in  which  I  work.  This  has  been  overcome 
to  a  great  degree  by  placing  soft  rubber  cushions  under  the  whole  appa- 
ratus."1 

I  have  recently  (1805)  seen  a  gaslight  which  I  believe  would  prove 
to  be  a  valuable  substitute  for  ordinary  street  gas,  and  I  judge  that, 
owing  to  its  superior  brilliancy,  a  single  jet  would  suffice  to  replace  the 
five  burners  in  a  linear  series  which  are  shown  in  the  above  figure. 
The  gas  referred  to  is  acetylene,  which  may  now  be  obtained  in  a 
liquid  form  in  strong  metal  cylinders.  Reference  has  already  been 
made  to  the  use  of  an  oil  light,  and  for  low  powers  an  ordinary  lamp 
with  a  flat  wick  may  be  used.  That  bacteria  may  be  successfully 
photographed,  with  an  amplification  of  one  thousand  diameters,  by 
means  of  an  oil  lamp  is  shown  by  the  beautiful  photomicrographs 
made  by  Capt.  W.  C.  Borden,  Assistant  Surgeon  U.  S.  Army. 
At  my  request  Dr.  Borden  has  prepared  the  following  detailed 
account  of  his  method  : 

'From  Johns  Hopkins  University  Circulars,  vol.  ix.,  No.  81,  p.  72. 


PHOTOGRAPHING   BACTERIA.  109 


DESCRIPTION     OF     APPARATUS     FOR     PHOTOMICROGRAPHY     BY     OIL 

LIGHT. 

The  apparatus  consists  of  a  camera,  hung-  in  a  vertical  position,  of  a 
microscope  with  substage  condensers,  suitable  objectives  and  projection 
oculars,  and  a  Laverne  tri-wick,  oil  stereopticon  with  the  projection  objec- 
tive removed. 

Tfie  Light. — After  trying  all  kinds  of  lamps,  I  found  that  the  best  illu- 
mination could  be  obtained  by  using  a  tri-wick  stereopticon  with  the  pro- 
jection objective  removed,  the  middle  wick  only  being  lighted.  The  large 
four-inch  condensers  serve  to  concentrate  the  light,  while  the  double  lantern 
body  prevents  the  radiation  of  heat  to  the  microscope  and  shuts  off  all  radiat- 
ing light.  Consequently  the  microscope  does  not  become  heated,  and  if  the 
room  is  darkened  the  absence  of  extraneous  light  greatly  aids  in  focussing  on 
the  camera  screen.  The  oil  light  itself  is  quite  yellow  and  so  nearly  mono- 
chromatic that  with  orthochromatic  plates  a  color  screen  is  seldom,  if  ever, 
required.  After  experimenting  by  taking  photographs  with  and  without  a 
screen,  I  have  found  110  particular  difference  in  result  even  when  photo- 
graphing difficult  bacteria,  and  now  seldom  use  one.  If  a  screen  is  used  a 
solution  of  bichromate  of  potash  and  sulphate  of  copper  in  dilute  ammonia 
water  placed  in  a  trough  between  the  lantern  and  microscope  gives  excellent 
results  and  does  not  materially  lengthen  the  time  of  exposure.  The  lantern 
is  placed  about  twelve  inches  in  front  of  the  microscope  and  with  its  central 
long  axis  in  a  plane  which  extends  through  the  centre  of  the  microscope 
mirror,  the  substage  condenser,  the  objective,  ocular  and  centre  of  camera. 

Microscope. — The  microscope  is  used  in  the  upright  position.  I  have 
used  this  position  rather  than  the  horizontal  for  several  reasons.  The 
microscope  is  used  on  the  work -table  in  an  upright  position,  and  in  working 
when  an  object  is  found  which  it  is  desired  to  photograph,  the  microscope 
without  changing  adjustments  has  only  to  be  carried  to  the  photomicro- 
graphic  apparatus,  placed  in  position,  correct  adjustments  of  light  made,  the 
camera  racked  into  contact  and  the  exposure  made.  With  a  conveniently 
placed  dark  room  the  whole  operation  will  occupy  but  a  few  minutes.  The 
upright  position  is  necessitated  when  liquid  preparations,  as  colonies  of 
bacteria  floating  on  liquefied  gelatin,  are  to  be  photographed,  or  when  the 
microscope  is  used  for  clinical  photomicrography,  as  in  photographing  uri- 
nary deposits  in  urine,  blood  corpuscles  in  Thoma  blood  counter,  etc.  In 
bacteriological  work  where  the  bacteria  are  stained  on  the  coyer  and  after 
mounting  the  balsam  is  not  quite  dry,  the  cover  is  apt  to  slip  if  the  micro- 
scope is  used  horizontally,  but  this  does  not  occur  with  the  microscope  placed 
vertically.  The  horizontal  position  and  long  extension  of  camera  is  neces- 
sary for  certain  work,  particularly  where  large  pictures  (i.e.,  over  four 
inches  in  diameter)  have  to  be  taken,  or  where  it  is  desired  to  obtain  high  am- 
plification by  extension  of  camera  rather  than  by  high  eyepiecing,  or  in 
photographing  test  diatoms  with  very  high  amplifications.  For  practical 
work,  however,  up  to  amplifications  of  one  thousand  diameters,  and  for 
photographs  for  illustration  or  reproduction,  which  are  seldom  required  of 
over  three  and  one-half  or  four  inches  in  diameter,  the  upright  position  is 
much  to  be  preferred  011  account  of  its  ease  of  application  and  its  practical 
advantages. 

Camera. — The  upright  position  of  the  microscope  necessitates  a  similar 
position  for  the  camera.  To  allow  easy  working,  the  camera  is  hung  on  a 
rack -work  attached  to  a  rigid  upright.  *  The  upright  is  placed  to  the  right  of 
the  microscope  so  that  it  will  be  out  of  the  way  while  working. 

Both  the  upper  and  the  lower  ends  of  the  camera  are  movable  on  the 
rack-work.  The  upper  end,  which  carries  the  screen  and  plate-holder,  is 
movable,  in  order  that  different  amplifications  within  limits  can  be  gotten 
with  the  same  objective.  The  lower  end  is  movable  that  it  may  be  racked 


110  PHOTOGRAPHING  BACTERIA. 

up  and  out  of  the  way  and  allow  the  operator  to  manipulate  the  microscope 
before  attaching  the  camera.  The  bellows  has  an  extension  of  two  feet, 
measured  from  the  eyepiece  of  the  microscope  to  the  focussing  screen.  This, 
with  a  two-millimeter  objective  and  projection  ocular  4,  gives  an  amplifica- 
tion of  one  thousand  diameters.  With  less  extension  of  bellows  and  lower 
objectives  amplifications  ranging  down  to  ten  diameters  may  be  obtained. 
In  focussing,  the  operator  can,  by  standing,  observe  the  image  on  the  screen 
with  a  focussing  glass  and  manipulate  the  fine  adjustment  of  the  microscope 
with  his  hand  without  using  a  focussing  r<xl,  though  a  suitable  focussing  rod 
can  be  easily  fastened  to  the  camera  upright  if  desired. 

Setting  Up  the  Apparatus. — The  camera  being  hung  on  the  rack-work, 
the  microscope  is  placed  beneath  it,  a  stage  micrometer  is  placed  on  the  stage 
and  a  medium-power  objective  and  eyepiece  attached  to  the  microscope. 
Light  is  reflected  from  the  lantern  upon  the  object  by  the  mirror  of  the 
microscope,  the  observer  accurately  centres  the  micrometer,  then  removing 
the  working  eyepiece  a  projection  ocular  is  inserted,  the  camera  racked 
down,  and  with  the  image  of  the  micrometer  projected  on  the  camera  screen 
the  microscope  is  moved  in  such  position  that  the  centre  of  the  micrometer 
image  is  exactly  in  the  centre  of  the  screen.  This  position  of  the  microscope 
is  marked  once  for  all,  and  whenever  afterward  the  microscope  is  placed  in 
the  same  place  the  centre  of  the  object  will  be  projected  on  the  centre  of  the 
screen.  To  correctly  place  the  lantern,  a  lower-power  objective  is  used,  to- 
gether with  a  high-power  (Abbe)  condenser.  The  objective  is  accurately 
focussed  on  the  lines  of  the  stage  micrometer;  by  adjusting  the  substage  con- 
denser a  clear  image  of  the  lamp  flame  is  projected  on  the  plane  of  the  ob- 
ject (micrometer)  and  the  lantern  is  moved  to  such  position  that  the  image 
will  be  central.  If  the  camera  is  attached,  the  image  will  appear  central 
on  the  focussing  screen. 

This  position  of  the  lantern,  like  that  of  the  microscope,  should  be  fixed. 

To  Photograph. — In  photographing  by  oil  light  with  all  but  the  lowest 
]x>wers  some  form  of  substage  condenser  is  necessary.  This  is  due  to  the  fact 
that  the  source  of  light  must  always  be  focussed  on  the  object  in  order  to  give 
proper  definition.  In  working  with  the  objectives  of  four  millimetres  or 
lower,  it  will  be  found  advantageous  to  use  objectives  of  lower  power  as 
substage  condensers,  for  it  will  be  found  that  if  placed  in  the  substage  for 
ordinary  work  they  greatly  improve  the  definition  of  objects.  In  fact  it  may 
be  laid  down  as  a  general  rule  that  whatever  with  a  given  light  gives  the 
best  definition  to  the  observer's  eye  will  give  the  sharpest  photographic  iinairr. 
Consequently,  in  high-power  work  where  a  condenser  is  used  it  will  seldom 
be  necessary  to  change  the  microscope  attachments  when  a  photograph  has 
to  be  taken;  for  in  bacteriological  work  the  Abbe  condenser  which  gives 
good  definition  will,  when  properly  adjusted,  give  good  photographic  defi- 
nition also,  statements  to  the  contrary  notwithstanding. 

To  photograph,  place  the  microscope  and  lantern  in  position,  light  the 
centre  wick  of  the  lamp,  place  a  ground  glass  between  the  lamp  and  camera, 
and  focus  the  objective  accurately  on  the  object.  The  ground  glass  is  used 
on  I  v  to  reduce  the  light  which  might  otherwise  injure  the  observer's  eye. 

The  ground  glass  is  then  removed,  a  fine  wire  screen  placed  close  against 
the  front  of  the  lantern  condenser,  and  by  means  of  the  substage  condenser 
an  imago  of  the  screen  is  projected  accurately  on  the  object.  This  is  very 
important,  for  it  is  necessary  that  the  light  should  be  accurately  focussed  on 
the  object  in  <  >rder  to  produce  sharp  definition.  After  focussing  the  light,  the 
screen  is  removed  and  an  opal  glass  is  put,  in  its  place.  On  looking  through 
the  eyepiece  a  clear  sharp  image  of  the  object  will  be  seen.  If  an  Abbe  con- 
denser is  used  the  iris  diaphragm  of  the  condenser  should  now  be  carefully 
opened  and  dosed  until  such  an  aperture  is  obtained  that  to  the  observer's 
eve  the  object  appears  to  the  best  advantage.  The  opal  glass  is  now  removed, 
the  camera  attached  to  the  microscope,  and  the  projected  image  focussed  on 
the  camera  screen,  preparatory  to  exposure. 


PHOTOGRAPHING  BACTERIA.  HI 

Plate  Used.—  Orthochromatic  plates  only  should  be  used.  Of  these  I  use 
the  Cramer  rapid,  isochromatic  plate  exclusively.  With  these  when  photo- 
graphing bacteria  and  using  an  amplification  of  one  thousand  diameters  the 
exposure  will  vary  from  one  and  one-half  to  three  minutes,  two  minutes 
being  about  the  average. 

It  is  with  these  plates  that  I  have  found  a  color  screen  unnecessary,  and 
since  using  them  I  have  had  no  difficulty  in  photographing  bacteria,  for  they 
are  particularly  sensitive  to  the  yellow-colored  oil  light. 

Possibly  other  makes  of  orthochromatic  plates  might  be  found  to  work 
equally  well,  but  the  oil  light  works  so  very  well  with  the  Cramer  isochro- 
matic that  I  have  had  no  desire  to  try  others. 

Development. — For  development,  I  have  obtained  best  results  with  for- 
mulas in  which  hydrochiiion  either  alone  or  with  some  other  developing 
agent  is  used.  The  following  gives  excellent  results,  and  I  prefer  it  to  other 
developers  as  it  gives  good  clear  negatives  of  sufficient  contrast  and 
gradation: 

No.  1. 

Water,       .......  10  ounces. 

Sodium  sulphite,        .  ....        1  ounce. 

Potassium  bromide,         .....  10  grains. 

Hydrochinon,  .  .  .  .  .  .30  grains. 

Metol,        .  .  .  .  .  .  40  grains. 

No.  2. 

Water,  .......      10  ounces. 

Sodium  carbonate,          ......         300  grains. 

Use  equal  parts  of  No.  1  and  No.  2. 

Development  should  be  continued  until  sufficient  density  is  obtained.  In- 
tensification should  be  rarely  required,  for  with  proper  exposure  and  develop- 
ment a  good  negative  can  usually  be  obtained.  If  intensification  is  neces- 
sary, after  fixing  and  washing  the  plate,  I  prefer  to  use  a  saturated  aqueous 
solution  of  bichloride  of  mercury,  followed  by  washing,  the  application  of 
dilute  ammonia  water,  and  a  final  washing. 

Students  who  desire  to  perfect  themselves  in  the  art  of  making 
photomicrographs  are  advised  to  first  make  themselves  familiar  with 
the  technique  of  photography  with  a  landscape  or  portrait  camera, 
and  not  to  undertake  the  more  difficult  task  of  photographing  bac- 
teria until  they  know  how  to  make  a  good  negative  and  to  judge 
whether  an  exposure  has  been  too  long  or  too  short,  etc. 


1'LATH  I. 

STERNBERG'S  BACTERIOL' 


3. 


*!&'   I 


4 


I'LATl-;   11. 

STERNBERG'S  BACTERIOLOGY 


• 


I'1  IK    a. 


4. 


I1' IK.   5. 


KM:.  <',. 


PLATE  I. 

PHOTOMICROGRAPHS  OP  BACTERIA  MADE  BY  GASLIGHT. 

FIG.  1. — Streptococcus  cadaveris,  from  a  culture  in  agua  coco;  stained 
with  f uchsin.  x  1,000.  (Steriiberg.) 

FIG.  2. — Streptococcus  Ha vaniensis.  x  1,000.  From  a  photomicrograph. 
(Sternberg.) 

FIG.  3. — Bacillus  cuniculicida  Ha  vaniensis,  from  peritoneal  cavity  of 
inoculated  rabbit,  showing  leucocytes  containing  bacilli  and  free  bacilli; 
stained  with  f  uchsin.  x  1,000.  (Sternberg.) 

FIG.  4.— Bacillus  cadaveris,  smear  preparation  from  yellow-fever  liver  kept 
for  forty-eight  hours  in  an  antiseptic  wrapping  (Havana,  1889) ;  stained  with 
fuchsin.  x  1,000.  (Sternberg.) 

Note. — All  of  the  above  photomicrographs  were  made  with  the  three- 
millimetre  apochromatic  horn.  ol.  im.  objective  and  projection  eye-piece  of 
Zeiss. 

PLATE  II. 

PHOTOGRAPHS  OF  COLONIES    (IN  ESMARCH  ROLL  TUBES)  AND  OF  TEST-TUBE 

CULTURES. 

FIG.  1. — Colonies  of  Bacillus  leporis  lethalis,  in  gelatin  roll  tube,  end  of 
forty-eight  hours  at  room  temperature,  x  5.  (Sternberg.) 

FIG.  2. —Colonies  of  Bacillus  coli  similis  in  gelatin  roll  tube,  end  of 
twenty-four  hours  at  22°  C.  x  10.  (Sternberg.) 

FIG.  3. — Stick  culture  of  Bacillus  coli  similis  in  nutrient  gelatin,  end  of 
seven  days  at  20°  C.  (Sternberg.) 

FIG.  4. — Stick  culture  of  Bacillus  intestinus  motilis  in  nutrient  gelatin, 
end  of  four  days  at  22°  C.  (Sternberg.) 

FIG.  5.— Stick  culture  of  Bacillus  leporis  lethalis  in  nutrient  gelatin,  end 
of  eight  days  at  22°  C.  (Sternberg.) 

FIG.  6. — Stick  culture  of  Micrococcus  tetragenus  versatilis  in  nutrient 
gelatin,  end  of  two  weeks  at  22 3  C.  (Sternberg.) 

FIG.  7.— Colonies  of  Bacillus  cuniculicida  Ha  vaniensis  in  gelatin  roll 
tube,  end  of  forty-eight  hours  at  21°  C.  x  10.  (Sternberg.) 

FIG.  8. — Colonies  of  Bacillus  coli  communis  in  gelatin  roll  tube,  end  of 
forty-eight  hours  at  22°  C.  x  10.  (Sternberg.) 


PART   SECOND. 


GENERAL  BIOLOGICAL  CHARACTERS: 

INCLUDING   AX   ACCOUNT   OF   THE   ACTION   OF   ANTISEPTICS 
AND   GERMICIDES. 

I.  STRUCTURE,   MOTIONS,  REPRODUCTION.     II.    CONDITIONS  OF  GROWTH. 

III.   MODIFICATIONS  OF  BIOLOGICAL  CHARACTERS.     IV.  PRODUCTS  OF 

VITAL  ACTIVITY.  V.  PTOMAINES  AND  TOXALBUMINS.    VI.  INFLUENCE 

OF  PHYSICAL  AGENTS.    VII.  ANTISEPTICS  AND  DISINFECTANTS 

— GENERAL  ACCOUNT  OF  THE  ACTION  OF.    VIII.  ACTION  OF 

GASES  AND  OF  THE  HALOID  ELEMENTS  UPON  BACTERIA. 

IX.  ACTION  OF  ACIDS  AND  ALKALIES.    X.  ACTION  OF 
VARIOUS  SALTS.  XI.  ACTION  OF  COAL-TAR  PRO- 
DUCTS,  ESSENTIAL  OILS,  ETC.     XII.  AC- 
TION OF  BLOOD  SERUM  AND  OTHER  OR- 
GANIC LIQUIDS.    XIII.  PRACTICAL 
DIRECTIONS  FOR  DISINFECTION. 


PART    SECOND. 


I. 
STRUCTURE,   MOTIONS,   REPRODUCTION. 

THE  bacteria  are  unicellular  vegetable  organisms,  and  consist  of 
a  cell  membrane  enclosing  transparent  and  apparently  structureless 
protoplasm.  The  very  varied  biological  characters  which  distin- 
guish different  species  make  it  evident,  however,  that  there  are  es- 
sential differences  in  the  living  cell  contents,  although  these  differ- 
ences are  not  revealed  by  our  optical  appliances.  And  among  the 
bacteria,  as  in  the  cells  of  higher  plants  and  animals,  the  peculiar 
biological  characters  of  a  species  are  transmitted  to  the  cellular  pro- 
geny of  each  individual  cell.  These  characters  are,  however,  sub- 
ject to  various  modifications  as  a  result  of  differing  conditions  of 
environment,  as  is  the  case  with  plants  and  animals  higher  in  the 
scale  of  existence,  and  in  this  way  more  or  less  permanent  varieties 
are  produced.  It  is  probable  that  among  these  lowly  plants  species 
are  evolved  more  quickly,  as  a  result  of  the  laws  of  natural  selec- 
tion, in  the  struggle  for  existence,  than  among  those  of  more  com- 
plex organization.  Still,  this  has  not  been  proved,  and,  on  the  ptljer 
hand,  we  have  ample  evidence  that  widely  distributed  species  exist 
having  very  definite  morphological  and  biological  characters  wnich 
enable  us  to  recognize  them  wherever  found. 

It  has  generally  been  supposed  that  these  simple  vegetable  cells 
are  destitute  of  a  nucleus,  but  a  recent  author  (Frankel)  suggests 
the  probability  that  a  nucleus  may  exist,  although  it  has  not;  been 
demonstrated.  This  suggestion  is  based  upon  the  fact  that  in  stain- 
ing bacteria  very  quickly  it  sometimes  happens  that  a  portion  of  the 
protoplasm  is  sharply  differentiated  by  taking  the  stain  more  deeply 
than  the  remaining  portion. 

Sjobring  in  1892  made  an  investigation  for  the  purpose  of 
ascertaining  the  structure  of  bacterial  cells.  Various  methods 
were  employed,  but  the  most  satisfactory  results  were  obtained  by 
fixing  with  nitric  acid,  with  or  without  alcohol,  and  without  pre- 


116  STRUCTURE,    MOTIONS,    REPRODUCTION. 

vious  drying  ;  the  preparations  were  then  stained  with  carbol-meth- 
ylene-blue  or  carbol-f  uchsin  solution  ;  they  were  decolorized  with 
nitric  acid  and  examined  in  glycerin  or  in  water.  By  this  procedure 
the  author  named  was  able  to  demonstrate  two  kinds  of  corpuscles. 
One  of  these  may  be  seen  just  inside  the  cell  wall ;  it  stains  deeply 
with  the  carbol-fuchsiii  solution.  The  other  lies  in  a  position  analo- 
gous to  that  occupied  by  the  nucleus  of  vegetable  cells  higher  in  the 
scale,  and  resembles  this  both  in  its  resting  condition  and  in  the 
process  of  indirect  division. 

In  his  address  before  the  International  Medical  Congress  of  Ber- 
lin (1890)  Koch  says  : 

"  We  had  not  succeeded,  in  spite  of  the  constantly  improving 
methods  of  staining  and  in  spite  of  the  use  of  objectives  with  con- 
stantly increasing  angles  of  aperture,  in  learning  more  with  reference 
to  the  interior  structure  of  the  bacteria  than  was  shown  by  the  origi- 
nal methods  of  staining.  Only  very  recently  new  methods  of  stain- 
ing appear  to  give  us  further  information  upon  the  structure  of  the 
bacteria,  inasmuch  as  they  serve  to  differentiate  an  interior  portion 
of  the  protoplasm,  which  should  probably  be  regarded  as  a  nucleus, 
from  an  exterior  protoplasmic  envelope  from  which  is  given  off  the 
organ  of  locomotion,  the  flagellum." 

Although  usually  transparent,  the  protoplasm  sometimes  presents 
a  granular  appearance.  The  botanist  Van  Tieghem  claims  to  have 
found  chlorophyll  grains  in  some  water  bacteria  studied  by  him,  and 
in  the  genus  Beggiatoa  grains  of  sulphur  are  found  embedded  in  the 
protoplasm  of  certain  species. 

The  cell  membrane  in  certain  species  appears  to  be  very  flexible, 
a--  may  be  seen  in  those  which  have  a  sinuous  motion.  It  is  not 
easily  recognized  under  the  microscope,  but  by  the  use  of  reagents 
which  cause  the  protoplasm  to  contract  may  be  demonstrated — e.g., 
by  iodine  solution.  Outside  of  the  true  cell  membrane  a  gelatinous 
envelope — so-called  capsule — is  sometimes  seen.  This  may  perhaps 
be,  as  claimed  by  some  authors,  nothing  more  than  a  jelly-like  thick- 
ruing  of  the  outer  layers  of  the  cell  wall.  This  jelly-like  material 
causes  the  cells  to  adhere  to  each  other,  forming  zoogloea  masses. 
I  n  some  cases  the  growth  upon  the  surface  of  a  culture  medium  is 
extremely  viscid ,  and  may  be  drawn  out  into  long  threads  when 
touched  with  a  platinum  needle,  owing  to  the  gebitinous  intercellular 
substance  by  which  the  cells  are  surrounded. 

There  is  but  little  more  to  be  said  of  the  structure  of  these  minute 
organisms,  except  to  mention  the  fact  that  the  motile  species  arc 
provided  with  slrn<l<-r.  whip-like  appendages  called  flagella.  The 
micrococci  in  general  are  not  endowed  with  the  power  of  executing 
spontaneous  movements,  and  they  are  not  provided  with  flagolla. 


STRUCTURE,    MOTIONS,    REPRODUCTION.  117 

Bat  recently  two  motile  species  have  been  described,  and  in  one  of 
these — Micrococcus  agilis  of  Ali-Cohen — the  presence  of  flagella  has 
been  demonstrated. 

Many  of  the  bacilli  and  spirilla  are  actively  motile,  and  the  pre- 
sence of  flagella,  which  has  long  been  suspected,  has  recently  been 
demonstrated  for  a  considerable  number  of  species  by  Lofner  and 
others. 

It  must  be  remembered  that  the  molecular  movement  which  is 
common  to  all  minute  particles  suspended  in  a  fluid  is  a  vibratory 
motion  in  situ,  which  does  not  change  the  relative  position  of  the 
moving  particles.  This  so-called  Brownian  movement  has  frequently 
been  mistaken  for  a  vital  motion,  as  has  also  the  movement  due  to 
currents  in  the  liquid  in  which  non-motile  organisms  are  suspended. 
The  latter  is  to  be  distinguished  by  the  fact  that  the  microorganisms 
are  all  carried  in  one  direction.  This  movement  due  to  a  current,  in 
connection  with  the  vibratory  Brownian  movement,  is  very  deceptive, 
and  it  is  often  hard  for  a  beginner  in  bacteriological  study  to  con- 
vince himself  that  what  he  sees  is  not  a  vital  movement.  But  in 
true  vital  movements  we  have  progression  in  different  directions,  and 
the  individual  microorganisms  approach  and  pass  each  other,  often 
in  a  most  vigorous  and  active  manner,  passing  entirely  across  the 
field  of  view  or  changing  direction  in  an  abrupt  way.  Sometimes 
the  motion  is  slow  and  deliberate,  the  bacillus  progressing  with. a  to- 
and-fro  motion,  as  if  propelled  by  a  trailing  flagellum  ;  or  it  may  be 
serpentine  when  the  moving  filament  is  flexible;  or  again  it  is 
a  darting  forward  motion  which  is  so  rapid  that  the  eye  can  scarcely 
follow  the  moving  body.  The  spirilla  have  a  rotary  movement  as 
well  as  a  progressive  one,  and  this  is  often  extremely  rapid.  Some- 
times bacilli  spin  around  with  a  rotatory  motion,  as  if  they  were  an- 
chored fast  to  a  fixed  point,  as  they  may  be  by  the  flagellum  being 
attached  to  the  slide  or  cover  glass.  Frequently,  in  a  pure  culture, 
the  individual  bacilli  may  be  seen  to  come  to  rest,  and,  after  an  inter- 
val of  repose,  to  dart  forward  again  in  the  most  active  way.  Or  we 
may  find,  on  examining  the  same  culture  at  different  times,  that 
upon  one  occasion  there  is  no  evidence  of  vital  movements,  and  on 
another  all  of  the  bacilli  are  actively  motile.  These  differences  de- 
pend upon  the  age  of  the  culture,  temperature  conditions,  etc. 

Reproduction  by  binary  division  is  common  to  all  of  the  bacte- 
ria, and  in  many  species  this  is  the  only  mode  of  reproduction  known. 
When  circumstances  are  favorable  for  rapid  multiplication  the  indi- 
vidual cells  grow  in  length,  and  a  constriction  occurs  in  the  middle 
transverse  to  the  long  diameter.  This  becomes  deeper,  and  after  a 
time  the  cell  is  completely  divided  into  two  equal  portions,  which 
again  divide  in  the  same  way.  Separation  may  be  complete,  or  the 


118  STRUCTURE,    MOTIONS,    REPRODUCTION. 

cells  may  remain  attached  to  each  other,  forming  chains  (strepto- 
cocci) or  articulated  filaments  (scheinfaden  of  the  Germans). 

The  bacilli  and  spirilla  divide  only  in  a  direction  transverse  to  the 
long  diameter  of  the  cells,  but  among  the  micrococci  division  may 
occur  either  in  one  direction,  forming  chains  ;  or  in  two  directions, 
forming  tetrads  ;  or  in  three  directions,  forming  "  packets"  of  eight 
or  more  elements.  The  staphylococci,  in  which  the  cells  do  not  re- 
main associated,  divide  indifferently  in  any  direction. 

The  rapidity  of  multiplication  by  binary  division  varies  greatly  in 
different  species,  and  in  the  same  species  depends  upon  conditions  re- 
lating to  the  culture  medium,  age  of  the  culture,  temperature,  etc. 
Under  favorable  conditions  bacilli  have  been  observed  to  divide  in 
twenty  minutes,  and  it  is  a  matter  of  common  laboratory  experience 
that  colonies  of  considerable  size  and  containing  millions  of  bacilli 
may  be  developed  from  a  single  cell  in  twenty-four  to  forty-eight 
hours.  A  simple  calculation  will  show  what  an  immense  number  of 
cells  may  be  produced  in  this  time  as  a  result  of  binary  division  oc- 
curring, for  example,  every  hour.  The  progeny  of  a  single  cell 
would  be  at  the  end  of  twenty-four  hours  16,777,220,  and  at  the  end 
of  forty-eight  hours  the  number  would  be  281,500,000,000. 

Some  of  the  earlier  observers  have  noted  the  presence  of  oval  or 
spherical  refractive  bodies  in  cultures  containing  bacilli ;  but  that 
these  were  reproductive  elements,  although  suspected,  was  not  de- 
monstrated until  a  comparatively  recent  date.  Pasteur  was  one  of 
the  first  to  point  out  the  fact  that  certain  bacteria  have  two  modes  of 
reproduction — by  fission  and  by  the  formation  of  endogenous  spores  ; 
but  the  first  careful  study  of  the  last-mentioned  method  was  made  by 
Koch  in  his  classical  study  of  the  anthrax  bacillus  (1878),  and  by 
Cohii,  who  studied  the  formation  of  spores  in  Bacillus  subtilis. 

These  reproductive  bodies  serve  the  same  purpose  in  the  preserva- 
tion of  species  as  the  seeds  of  higher  plants.  They  resist  desiccation 
and  may  retain  their  vitality  for  months  or  years  until  circumstances 
HIV  favorable  to  their  development,  when,  under  the  influence  of  heat 
;m<l  moisture,  they  reproduce  the  vegetative  form — bacillus  or  spiril- 
lum— with  all  of  its  biological  and  morphological  characters.  They 
are  composed  of  condensed  protoplasm  which  retains  the  vital  char- 
acters of  the  soft  protoplasm  of  the  mother  cell  from  which  it  has 
been  separated  ;  and  it  is  evident  that  whether  reproduction  occurs 
1>>  fission  or  by  the  formation  of  endogenous  spores,  the  protoplasm 
<  >f  the  cells  in  a  pure  culture  of  any  microorganism  is  simply  a  sepa- 
rated portion  of  the  protoplasm  of  the  progenitors  of  these  cells. 

Some  of  the  bacilli  grow  out  into  long  filaments  l>ef ore  the  forma- 
tion of  spores  occurs  ;  and  these  filaments  may  be  associated  in  bun- 
dies  or  intertwined  in  irregular  masses.  At  first  the  protoplasm  of  the 


STRUCTURE,    MOTIONS,    REPRODUCTION.  119 

filaments  is  homogeneous,  but  after  a  time  it  becomes  segmented, 
and  later  the  protoplasm  of  each  segment  becomes  condensed  into 
a  spherical  or  oval  refractive  body,  which  is  the  spore.  For  a  time 
these  are  retained  in  a  linear  position  by  the  cell  membrane  of  the 
filament  (Fig.  75,  a),  but  this  is  after  a  while  dissolved  or  broken 
up  and  the  spores  are  set  free.  In  liquid  cultures  they  sink  to  the 
bottom  as  a  pulverulent  precipitate,  and  upon  the  surface  of  a  solid 
medium  they  form  a  layer  which  is  usually  of  a  white  or  yellowish- 
white  color,  and  which,  when  examined  under  the  microscope,  in  old 
cultures  is  found  to  consist  almost  entirely  of  shining  spherical  or 
oval  bodies  which  do  not  stain,  by  the  ordinary  methods,  with  the 
aniline  colors.  While  many  of  the  bacilli  during  the  stage  of  spore 
formation  grow  out  into  long  filaments,  others  do  not,  and  one  or 
more  spores  make  their  appearance  in  rods  of  the  ordinary  length 
which  characterizes  the  species.  These  may  be  located  in  the  centre 
of  the  rod  or  at  one  extremity  (Fig.  75,  6).  It  sometimes  occurs 


c- 


Fia. 75. 

that  when  a  single  central  spore  is  formed  the  rod  becomes  very 
much  enlarged  in  its  central  portion,  assuming  a  spindle  shape  (Fig. 
75,  c);  or  one  extremity  may  be  enlarged,  producing  forms  such  as 
are  shown  in  Fig.  75,  d.  Some  of  the  smaller  spherical  spores  mea- 
sure less  than  0.5  yu  in  diameter,  but  they  are,  for  the  most  part, 
oval  bodies  having  a  short  diameter  of  0. 5  to  1  /<  and  a  long  diame- 
ter of  one  to  two  /<,  or  even  more.  They  are  enveloped  in  a  cellular 
envelope  which,  according  to  some  observers,  consists  of  two  layers 
— an  exosporium  and  an  endosporium. 

The  germination  of  spores  has  been  studied  by  Prazmowski, 
Brefeld,  and  others.  The  process  is  as  follows  :  By  the  absorption 
of  water  they  become  swollen  and  pale,  losing  their  shining,  refrac- 
tive appearance.  Later  a  little  protuberance  is  seen  upon  one  side 
or  at  one  extremity  of  the  spore,  and  this  rapidly  grows  out  to  form 
a  rod  which  consists  of  soft-growing  protoplasm  enveloped  in  a 
membrane  which  is  formed  of  the  endosporium  or  inner  layer  of  the 
cellular  envelope  of  the  spore.  The  outer  envelope,  or  exosporium, 
is  cast  off  and  may  be  seen  in  the  vicinity  of  the  newly  formed  rod 
(Fig.  76).  Sometimes  the  vegetative  cell  emerges  from  one  extrem- 


120  -IIMCirKK,    MOTIONS,    REPRODUCTION. 

ity  of  the  oval  spore,  as  shown  at  a,  Fig.  70,  arid  in  other  species  the 
exosporium  is  ruptured  and  the  bacillus  emerges  from  the  side.  as 
seen  at  b. 

The  considerable  resistance  of  these  endogenous  spores  to  desic- 
cation, to  heat,  and  to  various  chemical  agents  is  an  important  fact 
both  from  a  biological  and  from  a  hygienic  point  of  view,  and  will 
be  fully  considered  in  a  subsequent  chapter.  The  fact  that  certain 
bacilli  and  spirilla  do  not  withstand  a  temperature  of  80°  to  90°  C. , 
which  does  not  destroy  the  vitality  of  known  spores,  leads  to  the  in- 
ference that  they  do  not  form  similar  reproductive  bodies.  But  re- 
productive elements  of  a  different  kind  are  described  by  some  botan- 
ists as  being  produced  during  the  development  of  these  bacteria, 
and  also  of  the  micrococci.  These  are  the  so-called  arthrospores. 
In  the  process  of  binary  division  certain  cells  in  a  chain  may  be  ob- 
served to  be  somewhat  larger  than  others  and  to  refract  light  more 
strongly.  The  same  may  be  true  of  certain  cells  in  a  culture  in 
which  the  elements  are  not  united  in  chains.  These  cells  are  believed 


a-- 


FIG.  76. 

by  De  Bary  and  others  to  have  greater  resisting  power  to  desiccation 
than  the  remaining  cells  in  the  culture,  and  to  serve  the  purpose  of 
reproductive  elements. 

It  has  generally  been  supposed  that  spore  formation  is  most  likely 
to  occur  when  the  pabulum  for  supporting  the  growth  of  the  vegeta- 
tive form  is  nearly  exhausted.  But,  as  pointed  out  by  Frankel,  facts 
do  not  support  this  view,  as  many  species  form  spores  when  condi- 
tions are  most  favorable  for  a  continued  development.  An  abundant 
supply  of  oxygen  favors  the  formation  of  spores  in  aerobic  species, 
and,  in  some  instances  at  least,  the  temperature  has  an  important  in- 
fluence upon  spore  formation.  Thus  the  anthrax  bacillus  does  not 
form  spores  at  temperatures  below  20°  C.  or  above  42°  C. 

The  very  interesting  fact  has  been  demonstrated  by  Lehman  and 
by  Behring  that  a  species  which  usually  forms  spores  may  be  so 
modified  by  certain  influences  that  it  is  no  longer  capable  of  spore 
production,  and  that  such  an  asporogenous  variety  may  be  cultivated 
for  an  indefinite  time  without  showing  any  return  to  the  stage  of 
spore  formation.  This  was  effected  in  Behring's  experiments  by 
cultivating  the  anthrax  bacillus  in  a  medium  containing  some  agent 
detrimental  to  the  vitality  of  the  vegetative  cells,  but  not  in  suffi- 
cient quantity  to  restrain  their  development. 


STRUCTURE,    MOTIONS,    REPRODUCTION.  121 

The  chemical  composition  of  the  bacterial  cells  has  been  inves- 
tigated by  Nencki,  Brieger,  and  others.  Putrefactive  bacteria  culti- 
vated in  a  two-per-cent  solution  of  gelatin,  and  which  produced  an 
abundant  intercellular  substance  connecting  the  cells  in  zoogloea 
masses,  were  found  by  Nencki  to  have  the  following  composition  : 
Water,  84. 26  per  cent ;  solids,  5. 74  per  cent,  consisting  of  albumin 
87.46  per  cent,  fat  6.41,  ash  3.04,  undetermined  remnant  3.09. 
The  albuminous  substance,  according  to  Nencki,  is  not  precipitated 
by  alcohol,  and  differs  in  its  chemical  composition  from  other  known 
substances  of  this  class.  He  calls  it  mykoprotein  and  gives  the  fol- 
lowing as  its  chemical  composition  :  C,  52.33  percent;  H,  7.55  per 
cent ;  N,  14.75  per  cent.  It  contains  no  sulphur  and  no  phosphorus. 
The  spores  of  the  anthrax  bacillus,  according  to  Nencki,  do  not  con- 
tain mykoprotein,  but  a  peculiar  albuminous  substance  which  he 
calls  anthrax-protein.  Brieger  analyzed  a  gelatin  culture  of  Fried- 
lander's  bacillus,  with  the  following  result  :  Water,  84.2  per  cent ; 
solids,  5.8  per  cent,  containing  1.74  per  cent  of  fats.  After  removal 
of  the  fat  the  solids  gave  an  ash  of  30.13  per  cent  ;  this  contains  cal- 
cium phosphate,  magnesium  phosphate,  sodium  sulphate,  and  sodium 
chloride.  The  amount  of  nitrogen  in  the  dried  substance  after  re- 
moval of  the  fat  was  9. 75. 


II. 

CONDITIONS  OF  GROWTH. 

BACTERIA  only  grow  in  presence  of  moisture,  under  certain  condi- 
tions of  temperature,  and  when  supplied  with  suitable  pabulum.  As 
they  do  not  contain  chlorophyll,  they  cannot  assimilate  carbon  diox- 
ide, and  light  is  not  favorable  to  their  development. 

The  aerobic  species  obtain  oxygen  from  the  air  and  cannot  grow 
unless  supplied  with  it.  The  anaerobic  species,  on  the  other  hand, 
will  not  grow  in  the  presence  of  oxygen,  and  must  obtain  this  ele- 
ment, as  they  do  carbon  and  nitrogen,  from  the  organic  material 
which  serves  them  as  food. 

As  a  class  the  bacteria  are  supplied  with  nutriment  by  the  higher 
plants  and  animals,  the  dead  tissues  of  which  they  appropriate,  and 
which  it  is  their  function  to  decompose,  releasing  the  organic  ele- 
ments as  simple  compounds  which  may  again  be  assimilated  by  the 
chlorophyll-producing  plants. 

Water  is  essential  for  the  development  of  bacteria,  and  many  spe- 
cies have  their  normal  habitat  in  the  waters  of  the  ocean,  of  lakes, 
and  of  running  streams  ;  others  thrive  upon  damp  surfaces  or  in  the 
interior  of  moist  masses  of  organic  material.  Many  species  grow  in- 
differently either  in  salt  or  fresh  water,  but  it  is  probable  that  cer- 
tain species  will  be  found  peculiar  to  the  waters  of  the  ocean.  Some 
of  the  water  bacteria  multiply  in  the  presence  of  an  exceedingly 
minute  amount  of  organic  pabulum,  or  even  in  distilled  water.  This 
is  shown  by  the  experiments  of  Bolton  and  others.  The  author 
named  tested  two  species  of  water  bacteria  (Micrococcus  aquatilis 
,ind  Bacillus  erythrosporus)  in  the  following  manner:  Ten  cubic 
centimetres  of  distilled  water  in  a  test  tube  were  infected  with  a  si  MM  II 
quantity  of  a  culture  of  one  of  these  microorganisms.  A  drop  from 
this  tube  was  transferred  to  the  same  quantity  of  distilled  water  in 
a  second  tube,  and  from  this  to  a  third.  The  number  of  bacteria  in 
this  tube  No.  '.\  \VMS  now  ascertained  by  counting,  and  it  was  put 
a>i<lo  for  two  or  throe  days,  at  the  end  of  which  time  the  number  WMS 
M--MMI  estimated  by  counting.  In  every  case  there  was  an  enormous 
increase  in  the  number  of  bacteria.  In  order  to  be  sure  that  the  dis- 


CONDITIONS   OF   GROWTH.  123 

tilled  water  was  pure,  it  was  distilled  a  second  time  in  a  clean  glass 
retort,  but  the  result  was  the  same.  Bolton  remarks,  with  reference 
to  these  results:  "If  we  seek  to  explain  this  remarkable  fact  we 
must  remember,  in  the  first  place,  what  an  extremely  small  abso- 
lute mass  is  represented  by  an  enormous  number  of  bacteria,  and 
what  a  minute  amount  of  material  is  required  for  the  formation  of 
this  mass.  In  ten  cubic  centimetres  of  distilled  water,  in. the  experi- 
ment last  referred  to,  there  were  about  twenty  million  bacteria  (two 
million  per  cubic  centimetre).  If  we  estimate  the  diameter  of  each 
at  one  ^,  with  a  specific  weight  of  1,  the  absolute  weight  would 

be  for  the  entire  number  one-one-hundredth  of  a  milligramme 

that  is  to  say,  a  quantity  which  cannot  be  determined  by  any  of  our 
methods  of  weighing." 

Bolton  supposes  that  the  small  amount  of  organic  pabulum  re- 
quired fell  into  the  water  in  the  shape  of  dust,  or  was  attached  to  the 
walls  of  the  test  tube  in  spite  of  all  the  precautions  taken. 

Nitrogen  is  chiefly  obtained  from  albuminoid  substances,  but 
Pasteur  has  shown  that  it  may  also  be  obtained  from  ammonia. 
This  is  shown  by  cultivating  bacteria  in  a  medium  containing  an 
ammonia  salt,  as  in  the  following  : 

PASTEUR'S  SOLUTION. 

Distilled  water,               ......  100 

Cane  sugar,              ......  10 

Tartrate  of  ammonia,    ......  1 

Ashes  of  one  gramme  of  yeast,     ....  0.075 

CORN'S  SOLUTION. 

Distilled  water,  ......  100 

Tartrate  of  ammonia,  .....  1 

Ashes  of  yeast,  .  .  .  .  .  .  »  1 

Many  bacteria  multiply  abundantly  in  these  solutions. 

Carbon  is  obtained  from  the  various  organic  substances  contain- 
ing it ;  among  others,  from  starch,  sugars,  glycerin,  organic  acids 
and  their  salts,  etc. 

Temperature. — There  are  certain  limits  of  temperature  within 
which  development  may  take  place,  but  these  differ  greatly  with 
different  species.  As  a  rule,  growth  is  arrested  when  the  tempera- 
ture falls  below  10°  C.  (50°  F.),  but  some  species  multiply  at  a  still 
lower  temperature.  Thus  Bolton  observed  a  very  decided  increase 
in  certain  water  bacteria  kept  in  an  ice  chest  at  6°  C.,  and  other  ob- 
servers have  witnessed  development  at  the  freezing  temperature. 

Most  saprophytic  bacteria  grow  within  rather  wide  temperature 
limits,  but  the  rapidity  of  development  is  greatest  at  a  certain  favor- 
able temperature,  which  is  usually  between  25°  and  30°  C.  The 


124  CONDITIONS   OF   GROWTH. 

parasitic  species  have  a  more  restricted  ran^v.  which  approaches  the 
normal  temperature  of  the  animals  in  which  they  habitually  de- 
velop. At  40°  C.  (104°  F.)  growth,  as  a  rule,  ceases,  but  there  are 
some  notable  exceptions  to  this  rule. 

Miquel  some  years  ago  found  a  bacillus  in  the  water  of  the  Seine 
which  grew  at  a  temperature  of  69°  to  70°  C. ;  Van  Tieghem  reports 
having  observed  species  in  thermal  waters  capable  of  growth  at  a 
still  higher  temperature  (74°  C.)  ;  and  Globig  has  more  recently  ob- 
tained from  garden  earth  several  species  which  multiplied  at  65°  C. 
Some  of  the  species  found  by  the  last-named  observer  were  even 
found  to  require  a  temperature  of  about  60°  for  their  development; 
and  yet  this  temperature  is  quickly  fatal  to  a  large  number  of  the 
best  known  species. 

Lorv  temperatures,  while  arresting  the  growth  of  bacteria,  do  not 
destroy  their  vitality.  This  has  been  demonstrated  by  numerous  ex- 
periments, in  which  they  have  been  exposed  for  hours  in  a  refrigerat- 
ing mixture  at  —18°  C.  Frisch  has  even  subjected  them  to  a  tempe- 
rature of  —  87°  C.  by  the  evaporation  of  liquid  carbon  dioxide,  and 
found  that  they  still  grew  when  placed  in  favorable  conditions. 

Parasitism. — The  strict  parasites  grow  only  in  the  bodies  of  liv- 
ing animals,  or  in  artificial  media  kept  at  a  suitable  temperature. 
As  examples  we  may  mention  the  bacillus  of  tuberculosis,  the  bacil- 
lus of  leprosy,  the  micrococcus  of  gonorrhoea,  the  spirillum  of  re- 
lapsing fever.  There  is  also  a  large  class  of  facultative  para- 
sites which,  when  introduced  into  the  body  of  a  susceptible  animal, 
multiply  in  it,  and  may  continue  to  live  as  parasites  so  long  as  they 
are  transferred  from  one  animal  to  another,  but  which  are  also  able 
to  live  as  saprophytes  independently  of  a  living  host.  To  this  class 
belong  the  pus  cocci,  the  bacillus  of  typhoid  fever,  the  spirillum  of 
cholera,  and  many  others. 

It  seems  extremely  probable  that  the  strict  parasites  were  at  one 
time  capable  of  living  a  saprophytic  existence,  and  that  their  restric- 
tion to  a  parasitic  mode  of  life  has  been  effected  in  course  of  time  in 
accordance  with  the  laws  of  natural  selection.  This  view  is  sup- 
ported by  the  fact  that  the  tubercle  bacillus,  which  has  been  regarded 
as  a  strict  parasite,  which  can  only  be  cultivated  artificially  under 
very  special  conditions,  has  been  shown  \x>  be  capable  of  modification  in 
this  regard  to  such  an  extent  that  when  cultivated  for  a  time  in  a  favor- 
able medium — bouillon  with  five  per  cent  of  glycerin — it  will  even  grow 
in  ordinary  bouillon  made  from  the  flesh  of  a  calf  or  a  fowl  (Roux). 

licdcf i'>n  nf  Medium.      Sonic  bacteria  groxv  readily  in  a  medium 

having  an  acid  reaction,  while  the  slightest  trace  of  aridity  prevents 
the  development  of  others.  As  a  rule,  the  pathogenic  species  require 
a  neutral  «>r  slightly  alkaline  culture  medium. 


CONDITIONS   OF   GROWTH.  125 

While  many  species  grow  in  various  media  and  under  various 
conditions  of  temperature,  etc.^  others  are  greatly  restricted  in  this 
regard  ;  thus  Bumm  only  succeeded  in  cultivating  the  gonococcus 
upon  human  blood  serum,  and  even  upon  this  was  not  able  to 
carry  it  through  a  series  of  successive  cultures.  It  is  very  probable 
that  certain  species  can  only  grow  in  association  with  others  which 
elaborate  products  necessary  for  their  development. 

Substances  favorable  for  the  growth  of  a  particular  species  may 
restrain  its  development  if  present  in  too  large  an  amount.  Thus 
the  phosphorescent  bacilli  multiply  abundantly  in  a  nutrient  solution 
containing  2.5  per  cent  of  sodium  chloride  ;  but  this  amount  would 
restrain  the  development  of  some  other  species,  and  a  considerable 
increase  in  the  quantity  of  salt  prevents  the  growth  of  all  microor- 
ganisms. In  the  same  way  the  addition  of  two  per  cent  of  glucose 
to  culture  solutions  is  favorable  for  the  development  of  certain  spe- 
cies, and  especially  for  the  anaerobic  bacteria  ;  but  a  concentrated 
solution  of  the  same  substance  prevents  the  growth  of  all  bacteria. 

The  influence  of  one  species  upon  the  growth  of  another  has 
been  studied  by  various  bacteriologists,  and  especially  by  Sirotinin 
and  by  Freudeiireich.  When  several  species  are  associated  in  the 
same  culture  one  may  take  the  precedence  and  the  others  may  de- 
velop later  ;  or  two  or  more  species  may  develop  at  the  same  time  ; 
or  the  growth  of  one  species  may  prevent  the  development  of  an- 
other, either  (a)  by  exhausting  the  pabulum  necessary  for  its  growth 
or  (b)  by  producing  substances  which  inhibit  the  development  of  an- 
other species  or  destroy  its  vitality. 

Freudeiireich  found,  as  a  result  of  his  numerous  experiments, 
that  the  following  species  cause  a  change  in  bouillon  which  renders 
it  unfit  for  the  growth  of  other  species  :  Bacillus  pyocyaneus,  Bacil- 
lus cyanogenus,  Bacterium  phosphorescens,  Bacillus  prodigiosuf,  Spi- 
rillum cholera?  Asiaticne.  The  following  species  do  not  cause  such  a 
change  in  bouillon  as  to  render  it  unfit  for  the  growth  of  other  spe- 
cies :  Bacillus  typhi  abdomiiialis,  Bacillus  anthracis,  Bacillus  septi- 
caemias hsemorrhagicse,  Spirillum  tyrogeiium.  The  following  have  a 
decided  antagonism  :  Bacillus  pyogeiies  foetidus  prevents  the  growth 
of  Spirillum  cholera  Asiaticse  ;  Micrococcus  roseus  prevents  the 
growth  of  Micrococcus  tetragenus.  The  cholera  spirillum  will  not 
grow  in  sterilized  cultures  of  Bacillus  pyocyaneus,  or  in  bouillon 
which  has  served  for  a  previous  culture  of  the  same  microorganism 
(Kitasato).  Other  bacteria  which  fail  to  grow  in  bouillon  which 
has  already  served  for  the  cultivation  of  the  same  species  are  Bacil- 
lus typhi  abdominalis,  Bacillus  cyanogenus,  'Bacillus  prodigiosus, 
Micrococcus  roseus,  etc.  (Freudenreich). 


III. 

MODIFICATIONS  OF  BIOLOGICAL  CHARACTERS. 

WE  have  already  referred  to  the  production  of  an  asporogenous 
variety  of  the  anthrax  bacillus.  This  was  effected  by  Behring  by 
cultivation  in  media  containing  small  amounts  of  hydrochloric  acid, 
caustic  soda,  methyl  violet,  malachite  green,  and  various  other 
agents.  This  is  only  one  of  many  instances  of  a  change  in  biologi- 
cal characters  due  to  changed  conditions  of  environment.  We  have 
abundant  experimental  evidence  that  growth  may  occur  under  ad- 
verse conditions  when  the  species  is  gradually  habituated  to  these 
conditions.  Thus  the  temperature  limitations  may  be  passed  by  suc- 
cessive cultivations  at  temperatures  approaching  these  limits,  and 
bacteria  may  grow  in  the  presence  of  agents  which  in  a  given  pro- 
portion have  a  complete  restraining  influence  upon  their  develop- 
ment. For  example,  in  the  experiments  of  Kossiakoff,  published  in 
the  Annales  of  the  Pasteur  Institute  (vol.  i.),  it  was  found  that  the 
several  species  tested  all  became  habituated  to  the  presence  of  anti- 
septic agents  in  proportions  which  at  first  completely  restrained 
their  growth. 

This  modification  of  biological  characters  is  well  shown  in  the 
case  of  the  chromogenic  bacteria,  some  of  which  only  form  pig- 
ment under  exceptionally  favorable  conditions  of  growth.  It  has 
been  shown  by  several  observers  that  non-chromogenic  varieties 
of  some  of  the  best  known  chromogenic  species  may  be  produced 
by  special  methods  of  cultivation.  Thus  Wasserzug  obtained  a 
i  ion -chromogenic  variety  of  the  bacillus  of  green  pus  (Bacillus 
pyocyaneus)  by  the  action  of  time  added  to  that  of  antiseptics.  He 
Bays  :  "  These  two  actions  combined  have  permitted  me  to  obtain 
cultures  which  remained  without  color  in  a  durable  way,  and  in 
which,  consequently,  the  chromogenic  function  was  abolished  by 
heredity/'  In  the  case  of  a  chromogenic  bacillus  obtained  by  the 
writer  in  Havana  (my  Bacillus  Havanieiisis),  a  non-chromogenic  vari- 
ety was  obtained  from  a  culture  on  nutrient  agar  which  had  been  kept 
in  a  hermetically  sealed  glass  tube  for  about  a  year.  The  variety 
preserved  the  morphological  characters  of  the  original  stock,  but,  al- 


MODIFICATIONS   OF   BIOLOGICAL   CHARACTERS.  127 

though  carried  through  successive  cultures  for  a  considerable  period, 
did  not  regain  its  power  to  produce  the  brilliant  carmine  color  which 
is  the  most  striking  character  of  the  species.  Katz,  in  cultivating 
the  phosphorescent  bacilli  isolated  by  him  from  sea  water  at  New 
South  Wales,  found  that,  after  being  propagated  for  some  time  in 
artificial  media,  their  power  to  give  off  a  phosphorescent  light  was 
diminished  or  temporarily  lost.  He  also  found  that  two  species 
which  when  first  cultivated  did  not  liquefy  gelatin,  subsequently, 
after  a  year,  caused  liquefaction  of  the  usual  gelatin  medium. 

Modification  shown  in  Cultures. — When  bacteria  have  been 
subjected  to  the  action  of  heat  or  chemical  agents,  without  having 
their  vitality  completely  destroyed,  they  often  show  diminished  vigor 
of  growth.  Cultures  which  would  ordinarily  show  an  abundant  de- 
velopment within  twenty-four  hours  may  not  commence  to  grow  for 
several  days.  For  this  reason,  in  disinfection  experiments,  it  is  neces- 
sary to  test  the  question  of  destruction  of  vitality  by  leaving  the  cul- 
tures for  a  week  or  more  under  favorable  conditions  as  to  tempera- 
ture. In  plate  cultures  or  Esmarch  roll  tubes  a  few  colonies  may 
develop  in  this  tardy  way,  showing  that  there  was  a  difference  in  the 
vital  resisting  power  of  the  individual  cells,  some  having  survived 
while  the  majority  were  killed.  This  is  well  illustrated  by  Abbott's 
experiments  upon  the  germicidal  action  of  mercuric  chloride  as  tested 
upon  Staphylococcus  pyogenes  aureus.  Irregularities  in  the  results  in 
experiments  in  which  the  conditions  were  identical  having  been  no- 
ticed, Abbott  inferred  that  this  was  due  to  a  difference  in  the  resist- 
ing power  of  individual  cocci  (arthrospores  ?).  By  making  cul- 
tures from  colonies  which  developed  from  these  more  resistant  cocci, 
and  again  exposing  the  micrococci  in  these  cultures  to  mercuric  chlo- 
ride in  the  proportion  of  1:1,000  for  a  longer  time  and  making  new 
cultures  from  the  surviving  cocci,  and  so  on,  Abbott  obtained  cultures 
in  which  a  majority  of  the  cells  survived  exposure  to  a  solution  of  the 
strength  mentioned  for  ten  to  twenty  minutes,  whereas  in  his  original 
culture  most  of  the  cocci  were  killed  by  this  solution  in  five  minutes. 

These  changes  in  vital  resisting  power  enable  us  to  comprehend 
other  modifications  which  can  only  be  detected  by  chemical  or  bio- 
logical reactions.  Thus  the  reducing  power  for  various  substances 
may  be  modified  by  changes  in  the  conditions  of  environment.  And 
among  the  pathogenic  bacteria  changes  of  a  more  or  less  permanent 
nature  may  be  induced,  which  are  shown  by  a  modified  degree  of 
virulence  when  injected  into  susceptible  animals. 

Attenuation  of  Virulence  may  be  effected  by  several  methods, 
all  of  which  depend  upon  subjecting  the  cultures  to  prejudicial  in- 
fluences of  one  kind  or  another. 

Pasteur  first  announced,  in  1880,  that  the  microbe  of  fowl  cholera 


128  MODIFICATIONS   OF   BIOLOGICAL   CHARACTERS. 

could  be  modified  by  special  treatment  in  such  a  manner  that  it  no 
longer  produced  a  fatal  form  of  the  disease.  He  found  that  the  viru- 
lence was  greatest  when  cultures  were  made  from  fowls  which  had 
died  from  a  chronic  form  of  the  disease,  and  that  this  virulence  was 
not  lost  by  successive  cultivations  in  chicken  bouillon,  repeated  at 
short  intervals.  But  when  an  interval  of  more  than  two  months 
was  allowed  to  elapse  without  renewing  the  cultures,  the  virulence 
was  diminished  and  fewer  deaths  occurred  in  fowls  inoculated  with 
such  cultures.  This  diminution  of  virulence  became  more  marked 
in  proportion  to  the  length  of  time  during  which  a  culture  solution 
containing  the  microbe  remained  exposed  to  the  action  of  the  atmo- 
sphere, and  at  last  all  virulence  was  lost  as  a  result  of  the  death  of 
the  pathogenic  microorganism.  When  the  virus  was  preserved  in 
hermetically  sealed  tubes  it  did  not  undergo  this  modification,  but  re- 
tained its  full  virulence  for  many  months.  According  to  Pasteur, 
the  various  degrees  of  modification  of  virulence  resulting  from  pro- 
longed exposure  to  the  air  may  be  preserved  in  successive  cultures 
made  at  short  intervals.  Subsequent  experiments  with  cultures  of 
the  anthrax  bacillus  gave  similar  results  and  enabled  him  to  produce 
an  ' '  attenuated  virus  "  for  his  protective  inoculations. 

In  the  case  of  the  anthrax  bacillus  it  was  found  that  the  spores 
retain  their  full  virulence  for  years,  and  that  the  production  of  an  at- 
tenuated virus  required  the  exclusion  of  these  reproductive  elements. 
Cultivations  were  consequently  made  at  a  temperature  of  43°  to  43  ° 
C.,  at  which  point  this  bacillus  is  incapable  of  producing  spores. 
Cultivation  at  this  temperature  for  eight  days  gave  an  attenuated 
virus  suitable  for  use  in  protective  inoculations. 

Attenuation  by  Heat. — Toussaint  has  shown  that  a  similar  modi- 
fication of  virulence  may  be  produced  by  exposure  for  a  short  time 
to  a  temperature  a  little  below  that  which  destroys  the  vitality  of  the 
pathogenic  organism.  This  is  best  accomplished,  according  to  Chau- 
veau,  in  the  case  of  the  bacillus  of  anthrax,  by  exposure  for  eighteen 
minutes  to  a  temperature  of  50°  C.  Exposure  to  this  temperature  for 
twenty  minutes  is  said  to  completely  destroy  the  vitality  of  the  bacillus. 

Attenuation  by  Antiseptic  Agents. — The  writer,  in  1S80,  ob- 
tained evidence  that  attenuation  of  virulence  may  result  from  ex- 
IM  .sure  to  the  action  of  antiseptic  agents.  In  a  series  of  experiments 
made  to  determine  the  comparative  value  of  disinfectants,  the  blood 
of  a  rabbit  recently  dead  from  a  form  of  septicaemia  induced  by  the 
subcutaneous  injection  of  my  own  saliva,  and  due  to  the  presence  of 
a  micrococcus  (Micrococcus  pneumonue  crouposie),  was  subjected  to 
the  action  of  various  chemical  agents,  and  subsequently  injected 
into  a  rabbit  to  test  the  destruction  of  virulence.  In  the  published 
report  of  these  experiments  the  following  statement  is  made  : 


MODIFICATIONS    OF   BIOLOGICAL   CHARACTERS.  129 

1  'The  most  important  source  of  error,  however,  and  one  which 
must  be  kept  in  view  in  future  experiments,  is  the  fact  that  a  pro- 
tective influence  has  been  shown  to  result  from  the  injection  of  virus 
the  virulence  of  which  has  been  modified,  without  being  entirely  de- 
stroyed, by  the  agent  used  as  a  disinfectant." 

' '  Sodium  hyposulphite  and  alcohol  were  the  chemical  reagents 
which  produced  the  result  noted  in  these  experiments  ;  but  it  seems 
probable  that  a  variety  of  antiseptic  substances  will  be  found  to  be 
equally  effective  when  used  in  proper  proportion.  Subsequent  ex- 
periments have  shown  that  neither  of  these  agents  is  capable  of  de- 
stroying the  vitality  of  the  septic  micrococcus  in  the  proportion  used 
(one  per  cent  of  sodium  hyposulphite  or  one  part  of  ninety -five-per- 
cent alcohol  to  three  parts  of  virus),  and  that  both  have  a  restraining 
influence  upon  the  development  of  this  organism  in  culture  fluids."  ' 

Cultivation  in  the  Blood  of  an  Immune  Animal. — It  has 
been  shown  by  the  experiments  of  Ogata  and  Jasuhara  that  when 
the  anthrax  bacillus  is  cultivated  in  the  blood  of  an  immune  animal, 
such  as  the  dog  or  the  white  rat,  its  pathogenic  power  is  modified 
so  that  it  no  longer  kills  susceptible  animals  and  may  be  used  as  a 
vaccine. 

Pasteur  had  previously  shown  (1882)  that  the  virus  of  rouget  can 
be  attenuated  by  passing  it  through  rabbits. 

Recovery  of  Virulence. — Pasteur  has  shown  that  when  the  viru- 
lence of  a  pathogenic  organism  has  been  modified  it  may  be  re- 
stored by  successive  inoculations  into  susceptible  animals.  Thus  in 
the  case  of  the  anthrax  bacillus  a  culture  which  would  not  kill  an 
adult  guinea-pig  may  be  inoculated  into  a  very  young  animal  of  the 
same  species  with  a  fatal  result  ;  and  by  inoculating  the  blood  of 
this  animal  into  another,  and  so  on,  the  original  virulence  may  be 
restored,  so  that  a  culture  is  obtained  which  will  kill  a  sheep.  In 
the  same  way  the  attenuated  virus  of  fowl  cholera  may  be  restored 
to  full  vigor  by  inoculating  a  small  bird— sparrow  or  canary— to 
which  it  is  fatal.  After  several  successive  inoculations  the  virus 
resumes  its  original  activity. 

In  general,  pathogenic  virulence  is  increased  by  successive  inocu- 
lations into  susceptible  animals,  and  diminished  by  cultivation  in  arti- 
ficial media  under  unfavorable  conditions.  Thus  various  pathogenic 
bacteria  which  have  been  cultivated  in  laboratories  for  a  length  of 
time  are  likely  to  disappoint  the  student  if  he  makes  inoculation  ex- 
periments for  the  purpose  of  demonstrating  their  specific  action  as 
described  in  text  books. 

1  Quoted  from  "  Bacteria,"  pages  207,  208,  written  in  1883. 


IV. 
PRODUCTS  OF  VITAL  ACTIVITY. 

ALL  living  cells,  animal  or  vegetable,  while  in  active  growth, 
appropriate  certain  elements  for  their  nutrition  from  the  pabulum 
with  which  they  are  supplied,  and  at  the  same  time  excrete  certain 
products  which,  in  some  cases  at  least,  it  is  their  special  function  to 
produce.  In  the  higher  plants  and  animals  specialized  cells  excrete 
substances  which  are  injurious  to  the  economy  of  the  individual, 
and  secrete  substances  which  are  required  to  maintain  its  existence. 
As  an  example  in  animals  we  may  mention  the  excretion  of  urea  by 
the  epithelium  of  the  kidneys,  the  retention  of  which  is  fatal  to  the 
individual,  and  the  gastric  secretion  which  is  essential  for  its  con- 
tinued existence.  Among  the  higher  plants  we  have  an  immense 
variety  of  substances  formed  in  the  cell  laboratories,  some  of  which 
are  evidently  useful  for  the  preservation  of  the  species,  while  others 
are  perhaps  to  be  considered  simply  as  excretory  products.  The 
odorous  volatile  products  given  off  by  flowers  are  supposed  to  be 
useful  to  the  plant  in  attracting  insects  by  which  cross-fertilization 
is  effected.  The  various  poisonous  substances  stored  up  in  leaves 
and  bark  may  serve  to  protect  the  plant  from  enemies,  etc. 

The  minute  plants  with  which  we  are  especially  concerned  also 
produce  a  great  variety  of  substances,  some  of  which  may  be  useful 
to  the  species  in  the  struggle  for  existence.  Thus  the  deadly  pto- 
maines produced  by  some  of  the  pathogenic  bacteria  serve  to  para- 
lyze the  vital  resisting  power  of  living  animals  and  enable  the  para- 
sitic invader  to  thrive  at  the  expense  of  its  host.  In  the  present 
section  we  shall  consider  in  a  general  way  these  various  products  <  >t 
bacterial  growth. 

Pigment  Production. — A  considerable  number  of  species  arc 
distinguished  by  the  formation  of  pigment  of  various  colors  and 
shades.  We  have  all  of  the  shades  of  the  spectrum  from  violet  to 
red.  The  color,  as  a  rule,  is  only  produced  in  the  presence  of  oxy- 
gen, and  when  the  pigment-producing  microorganisms  are  massed 
upon  the  surface  of  a  solid  culture  medium  the  pigment  production 
is  often  limited  to  the  superficial  portion  of  the  mass.  In  some 
cases  a  soluble  pigment  is  formed  which  is  absorbed  by  the  transpa- 


ST  KR N 1 5 KK  (V  S  B  A CTE  R I  OLD  GY 


PIa^e  HI. 


Fig.l. 


2. 


3. 


fig.  4-. 


fig.] .  Sarcinalutea,a^ar  culture 

Fig. 2.  Bacillus  prodi^iosus,  a^ar  culture. 

Fig. 3.  Bacillus  pyocyanus,  agar  culture. 

Fiq.4".  Bacillus  Havamensis, potato  culture. 


PRODUCTS   OF   VITAL   ACTIVITY.  131 

rent  culture  medium,  coloring  especially  the  upper  portion,  in  stick 
cultures  in  nutrient  gelatin  or  agar.  This  is  the  case  with  Bacillus 
pyocyaneus,  .which  produces  a  blue  pigment  which  has  been  isolated 
and  carefully  studied  by  Gessard  and  others.  The  pigment,  which 
is  called  pyocyanin,  is  soluble  in  chloroform  and  crystallizes  from  a 
pure  solution  in  long  blue  needles.  Acids  change  the  blue  color  to 
red,  reducing  substances  to  yellow.  It  resembles  the  ptomaines  in 
its  chemical  reactions,  being  precipitated  by  platinum  chloride  and 
phosphomolybdic  acid. 

In  some  media  the  color  produced  by  the  Bacillus  pyocyaneus 
(bacillus  of  green  pus)  is  a  fluorescent  green.  The  recent  studies  of 
Gessard  show  that  this  is  a  different  pigment.  According  to  this 
author,  cultures  in  a  two-per-cent  solution  of  peptone  give  a  beautiful 
blue  tint,  the  production  of  which  is  hastened  by  adding  to  the  liquid 
five  per  cent  of  glycerin.  In  nutrient  gelatin  and  agar  cultures  a 
Fluorescent  green  color  is  developed,  which,  according  to  Gessard, 
is  due  to  the  presence  of  albumin.  Peptone  and  gelatin  are  said  to 
produce  pyocyanin  without  the  fluorescent-green  pigment,  and  cul- 
tures in  bouillon  to  give  both  this  and  pyocyanin.  In  milk  the 
fluorescent-green  color  is  first  seen,  but  subsequently,  when  the  ca- 
sein has  been  peptonized  by  a  diastase  produced  in  the  culture,  pyo- 
cyanin is  also  formed.  Several  other  microorganisms  are  known 
which  produce  a  fluorescent-green  color,  due  probably  to  the  same 
pigment  as  is  produced  by  the  bacillus  of  green  pus  in  albuminous 
media. 

Babes  claims  to  have  obtained  two  pigments  from  cultures  of  the 
Bacillus  pyocyaneus  in  addition  to  pyocyanin :  one,  soluble  in  alcohol, 
has  by  transmitted  light  a  chlorophyll-green  color,  by  reflected  light 
it  is  blue;  the  other,  insoluble  in  alcohol  and  chloroform,  by  trans- 
mitted light  is  of  a  dark  orange-red,  by  reflected  light  a  greenish- 
blue. 

In  Gessard's  latest  publication  (1891)  he  shows  that  the  produc- 
tion of  pyocyanin  or  of  the  fluorescent-green  pigment  does  not  de- 
pend alone  upon  the  culture  medium,  but  that  there  are  different 
varieties  of  the  Bacillus  pyocyaneus.  He  has  succeeded  in  producing 
four  distinct  varieties — one  which  produces  both  pyocyanin  and 
fluorescence,  one  which  produces  pyocyanin  alone,  one  which  pro- 
duces the  fluorescent-green  pigment  alone,  and  one  which  produces 
no  pigment.  The  last-mentioned  non-chromogenic  variety  was  pro- 
duced by  subjecting  the  second  variety  to  the  action  of  heat.  A 
temperature  of  57°  maintained  for  five  minutes  destroyed  the  power 
to  produce  pigment  without  destroying  the  vitality  of  the  bacillus, 
which  was  propagated  through  successive  cultures  without  regaining 
this  power. 


132  PRODUCTS   OF   VITAL   ACTIVITY. 

The  well-known  Bacillus  prodigiosus  (also  described  as  a  micro- 
coccus)  produces  a  red  pigment  which  is  insoluble  in  water  but  solu- 
ble in  alcohol.  By  the  addition  of  an  acid  the  color  becomes  car- 
mine and  then  violet,  which  is  changed  to  yellow  by  an  alkali.  The 
color  is  said  by  Schottelius  to  be  diffused  in  the  young  cells,  and 
after  the  death  of  the  cells  to  be  present  in  their  vicinity  in  the  form 
of  granules.  The  same  author  has  shown  that  by  subjecting  the 
bacillus  to  special  conditions  a  variety  may  be  obtained  which  no 
longer  produces  pigment. 

The  conditions  which  govern  the  formation  of  pigment  in  the 
chromogenic  bacteria  are  determined  with  comparative  facility  be- 
cause the  results  of  changed  conditions  are  apparent  to  the  eye  ;  in 
the  case  of  products  which  are  not  colored  the  difficulties  attending 
the  study  of  these  conditions  are  much  greater,  but  the  results  are  in 
many  instances  more  important.  The  following  are  among  the  best 
known  pigment-producing  (chromogenic)  bacteria  : 

Staphylococcus  pyogenes  aureus  (No.  1),  Staphylococcus  pyc- 
genes  citreus  (No.  3),  Sarcina  aurantiaca  (No.  226),  Sarcina  lutea 
(No.  227),  Bacillus  cyanogenus  (No.  257),  Bacillus  janthinus  (No. 
207),  Bacillus  fluorescens  liquefaciens  (No.  277),  Bacillus  indicus  (-No. 
283),  Bacillus  pyocyaneus  (No.  95),  Bacillus  prodigiosus  (No.  284), 
Spirillum  rubrum  (No.  429). 

Liquefaction  of  Gelatin. — Many  species  of  bacteria,  when 
planted  in  a  medium  containing  gelatin,  cause  a  liquefaction  of  the 
gelatin  in  the  immediate  vicinity  of  the  growing  microorganisms, 
while  many  others  multiply  abundantly  in  the  same  medium  with- 
out liquefying  the  gelatin.  This  character,  as  first  shown  by  Koch, 
is  an  important  one  in  the  differential  diagnosis  of  species  which  re- 
semble each  other  in  form  and  in  other  respects.  It  has  no  relation 
to  pathogenic  power,  as  some  liquefying  organisms  are  harmless  sap- 
rophytes and  some  deadly  disease  germs,  while,  on  the  other  hand, 
non-liquefying  bacteria  may  be  very  pathogenic  or  quite  innocent. 

Liquefaction  is  produced  by  a  soluble  peptonizing  ferment  formed 
during  the  growth  of  the  cells.  This  is  shown  by  the  fact  that  if  a 
liquefying  organism  is  cultivated  in  bouillon  and  the  living  cells  re- 
moved by  filtration  or  killed  by  heat,  the  power  of  liquefying  gelatin 
remains  in  the  culture  fluid.  This  was  first  observed  by  Bitter  (1880) 
and  independently  by  the  writer  in  1887.  In  experiments  made  to 
determine  the  thermal  death-point  of 'various  bacteria  the  writer 
found  that  when  cultures  of  liquefying  species  were  subjected  to  a 
temperature  which  killed  the  microorganisms,  a  few  drops  of  tin* 
culture  added  to  nutrient  gelatin  which  had  been  liquefied  by  heat 
prevented  it  from  subsequently  forming  a  solid  jelly  when  cold. 

In  a  study  of  the  ferments  produced  by  bacteria  which  cause 


PRODUCTS   OF   VITAL   ACTIVITY.  133 

liquefaction  of  gelatin — "tryptic  enzymes" — made  by  Fermi,  in  the 
laboratory  of  the  Hygienic  Institute  of  Munich  (1891),  the  following 
results  were  obtained : 

The  enzymes  were  not  obtained  pure,  and  their  isolation  from 
other  proteids  present  in  the  cultures  was  found  to  be  attended  with 
great  difficulties,  but  their  ferment  action  was  studied  and  was  found 
to  be  influenced  by  various  conditions. 

All  were  destroyed  by  a  temperature  of  70°  C. ,  but  the  enzymes 
produced  by  various  liquefying  bacteria  differed  considerably  as  to 
the  temperature  which  they  were  able  to  withstand.  Some  were  de- 
stroyed by  a  temperature  of  50°  to  55°  C. — Bacillus  megatherium, 
Bacillus  ramosus,  Staphylococcus  pyogenes  aureus  ;  some  by  a  tem- 
perature of  55°  to  60°  C. — Bacillus  subtilis,  Bacillus  pyocyaneus,  Ba- 
cillus fluorescens  liquefaciens,  Sarciua  aurantiaca;  some  by  65°  to 
70°  C. — Bacillus  anthracis,  Spirillum  cholera?  Asiaticse,  Spirillum  of 
Finkler  and  Prior,  Spirillum  tyrogenum. 

These  enzymes,  like  the  previously  known  pepsin,  trypsin,  and 
invertin,  do  not  dialyze. 

Only  a  few  of  these  bacteria  enzymes  acted  upon  fibrin,  and  no 
action  was  observed  upon  casein  or  upon  egg  albumen. 

Their  liquefying  action  upon  gelatin  was  prevented  by  the  action 
of  sulphuric  acid,  and  to  a  less  degree  by  nitric  acid,  but  was  not  in- 
terfered with  by  acetic  acid. 

The  liquefying  bacteria,  as  a  rule,  only  produce  enzymes  when 
cultivated  in  a  medium  containing  albumen. 

These  enzymes  are  not  produced  by  a  solution  of  the  protoplasm 
of  dead  bacterial  cells,  but  are  a  product  of  the  vital  activity  of  liv- 
ing cells. 

Among  the  numerous  liquefying  bacteria  known  to  bacteriolo- 
gists we  may  mention  the  following  species  as  deserving  the  student's 
special  attention  :  Staphylococcus  pyogenes  aureus  (No.  1),  Staphylo- 
coccus pyogenes  albus  (No.  2),  Sarcina  lutea  (No.  227),  Sarcina  au- 
rantiaca (No.  226),  Bacillus  anthracis  (No.  45),  Bacillus  pyocyaneus 
(No.  95),  Bacillus  subtilis  (No.  379),  Bacillus  indicus  (No.  283),  Ba- 
cillus prodigiosus  (No.  284),  Spirillum  cholera?  Asiatics  (No.  155), 
Spirillum  of  Finkler  and  Prior  (No.  156),  Proteus  vulgaris  (No.  97). 

Production  of  Acids. — Numerous  bacteria  give  an  acid  reaction 
to  the  media  in  which  they  are  cultivated,  and  the  acids  produced 
are  various — lactic,  acetic,  butyric,  propionic,  succinic,  etc. 

The  power  to  produce  an  acid  is  well  shown  by  adding  to  neu- 
tral or  alkaline  culture  media  a  solution  of  litmus.  The  change  in 
color  due  to  the  formation  of  an  acid  may  be  followed  by  the  eye, 
and  comparative  tests  may  be  made  to  aid  in  the  differentiation  of 
similar  bacteria. 


134  PRODUCTS   OF   VITAL   ACTIVITY. 

A  considerable  number  of  bacteria  are  able  to  produce  lactic 
acid  from  milk  sugar  and  other  carbohydrates.  One  of  these  is 
considered  the  special  lactic-acid  ferment — Bacillus  acidi  lactici — and 
is  the  usual  cause  of  the  acid  fermentation  of  milk.  Pure  cultures 
of  this  bacillus  introduced  into  sterilized  milk  or  solutions  of  milk 
sugar,  cane  sugar,  dextrin,  or  mannite,  give  rise  to  the  lactic-acid 
fermentation,  in  which  carbonic  acid  is  also  set  free.  The  proc-ess 
requires  free  access  of  oxygen,  and  progresses  most  favorably  at  a 
temperature  of  35°  to  40°  C.,  ceasing  at  about  45°.  In  milk,  coagu- 
lation of  the  casein  occurs  within  fifteen  to  twenty-four  hours  after 
adding  a  small  quantity  of  a  pure  culture  of  the  lactic-acid  bacillus. 
This  is  not  due,  however,  to  the  acid  fermentation,  but  to  a  ferment 
resembling  that  of  rennet,  which  is  produced  by  many  different 
bacteria,  some  of  which  do  not  produce  an  acid  reaction  of  the  milk. 
Among  the  bacteria  which  produce  lactic  acid  from  milk  sugar  we 
may  mention  the  staphylococci  of  pus,  Bacillus  lactis  aerogenes,  and 
Bacillus  coli  communis. 

The  formula  showing  the  transformation  of  sugar  into  lactic 
acid  is  usually  stated  as  follows  :  C8HjaO0  =  2(HC3H5O3). 

Acetic  acid  is  also  produced  from  dilute  solutions  of  alcohol  by 
the  action  of  a  special  bacterial  ferment,  which  accumulates  upon 
the  surface  of  the  fluid  as  a  mycoderma,  consisting  almost  entirely 
of  the  Bacillus  aceticus  (Mycoderma  aceti).  Free  access  of  oxygen 
is  required,  and  a  temperature  of  about  33°  C.  is  most  favorable  to 
the  process.  According  to  Duclaux,  the  "  Mycoderma  aceti "  oxi- 
dizes the  alcohol,  in  solutions  containing  it,  so  long  as  any  is  present, 
and  when  it  is  exhausted  it  oxidizes  the  acetic  acid  previously 
formed  by  oxidation  of  the  alcohol,  producing  from  it  carbon  diox- 
ide and  water. 

The  formation  of  acetic  acid  from  alcohol  is  shown  by  the  follow- 
ing formula  :  Ethyl  alcohol  CH3.CHa.OH  +  O2  =  CH8.COOH  +  H,O. 

Butyric  acid  is  produced  by  a  considerable  number  of  bacteria, 
one  of  which,  named  Bacillus  butyricus,  has  received  the  special  at- 
tention of  Prazmowski.  This  is  strictly  anaerobic.  In  solutions  of 
M.ireh,  dextrin,  sugar,  or  salts  of  lactic  acid,  when  oxygen  is  ex- 
cluded it  produces  butyric  acid  in  considerable  quantity,  and  at  the 
•ame  time  carbon  dioxide  and  hydrogen  gas  are  set  free.  Duclaux 
gives  the  following  formula  of  a  solution  containing  lactate  of  lime 
in  which  the  action  of  the  butyric-acid  ferment  may  be  well  studied  : 

Water,  .  .  .  .  .  8  to  10  litres. 

Lactate  of  lime  (pure),    ....  225  grammes. 

Phosphate  of  ammonia,          ....  0.75 

Phosphate  of  potash,.      .  .  .  .  0.4 

Sulphate  of  magnesa,  ....  0.4 

Sulphate  of  ammonia,      ....  0.2 


PRODUCTS   OF   VITAL   ACTIVITY. 


135 


This  is  introduced  into  a  flask  with  two  necks,  such  as  is  shown 
in  Fig.  77.  Having  filled  the  flask  with  the  culture  liquid,  the  bent 
neck  is  dipped  into  a  porcelain  dish  containing  the  same.  Heat  is 
then  applied  both  to  flask  and  dish,  and  the  liquid  in  each  is  kept  in 
ebullition  for  half  an  hour.  By  this  means  the  air  is  completely 
driven  out  of  the  flask.  This  is  now  allowed  to  cool,  while  the  fluid 
in  the  shallow  dish  is  kept  hot,  so  that  the  liquid  mounting  from  it 
into  the  flask  shall  be  free  from  air.  When  the  flask  is  full  it  is 
transferred  to  an  incubating  oven  heated  to  25°  to  30°  C.,  and  the  bent 
tube  is  immersed  in  a  dish  containing  mercury.  The  little  funnel 
attached  to  the  upright  tube  is  then  filled  with  carbon  dioxide  and  a 
culture  of  the  butyric-acid  bacillus  is  introduced  into  the  funnel. 
By  turning  the  stopcock  in  the  upright  tube  a  little  of  the  culture  is 


FIG.  77. 

admitted  to  the  flask  without  admitting  any  air.  Fermentation  com- 
mences very  soon,  as  is  seen  by  the  bubbles  of  gas  given  off.  The 
liquid  loses  its  transparency  and  the  lactic  acid  is  gradually  con- 
sumed, butyrate  of  lime  taking  the  place  of  the  lactate. 

Aerobic  bacilli  capable  of  producing  butyric  acid  in  culture  solu- 
tions containing  grape  sugar  or  milk  sugar  have  also  been  described 
by  Liborius  and  by  Hueppe. 

Fitz  has  shown  that  in  culture  solutions  containing  glycerin  the 
Bacillus  pyocyaneus  produces  butyric  acid  in  addition  to  ethyl  alcohol 
and  succinic  acid.  Bacillus  Fitzianus  also  produces  some  butyric  acid 
in  solutions  containing  glycerin,  although  the  principal  product  of  the 
fermentation  caused  by  this  microorganism  is,  according  to  Fitz, 
ethyl  alcohol,  twenty-nine  grammes  of  which  may  be  obtained  from 
one  hundred  grammes  of  glycerin. 


136  PRODUCTS   OF   VITAL  ACTIVITY. 

Botkin  (1892)  has  described  a  "Bacillus  butyricus"  (No.  40*,) 
which  he  has  not  been  able  to  identify  positively  with  the  butyric- 
acid  ferment  described  by  Prazmowski.  It  is  a  widely  distributed 
anaerobic  bacillus,  which  he  was  able  to  obtain  from  milk  or  water 
containing  it  by  placing  it  in  the  steam  sterilizer  for  half  an  hour. 
The  spores  resisted  this  temperature  and  subsequently  grew  in  anae- 
robic cultures,  in  a  suitable  medium,  while  all  other  bacteria  and 
spores  present  were  destroyed. 

The  writer  has  described  a  bacillus  which  causes  active  acid 
fermentation  in  culture  solutions  containing  glycerin.  The  aoid 
formed  is  volatile  and  is  probably  propionic  acid — see  Bacillus  acidi- 
formans. 

The  Caucasian  milk  ferment — Bacillus  Kaukasicus — produces 
a  variety  of  products  in  the  fermented  milk  which  is  a  favorite 
drink  among  the  Caucasians.  The  principal  ones  are  ethyl  alcohol, 
lactic  acid,  and  carbon  dioxide,  but  in  addition  to  these  small  quanti- 
ties of  succinic,  butyric,  and  acetic  acids  are  formed.  The  inhabi- 
tants of  the  Caucasian  mountains  prepare  this  fermented  drink  in  a 
very  simple  manner  from  the  milk  of  cows  or  goats,  to  which  they 
add  the  dried  ferment  collected  from  a  receptacle  in  which  the  fermen- 
tation had  previously  taken  place.  Fliigge  gives  the  following  di- 
rections for  the  preparation  of  this  drink  : 

"  Two  methods  may  be  employed.  In  the  first  the  dry  brown  kefir-kdr- 
ner  of  commerce  are  allowed  to  lie  in  water  for  five  to  six  hours  until  they 
swell;  they  are  then  carefully  washed  arid  placed  in  fresh  milk,  which 
should  be  changed  once  or  twice  a  day  until  the  korner  become  pure  white 
in  color  and  when  placed  in  fresh  milk  quickly  mount  to  the  surface — in 
twenty  to  thirty  minutes.  One  litre  of  milk  is  then  poured  into  a  flask  and  a 
full  tablespoonful  of  the  prepared  korner  added  to  it.  It  is  allowed  to  stand 
open  for  five  to  eight  hours;  the  flask  is  then  closed  and  kept  at  18°  C.  It 
should  be  shaken  every  two  hours.  At  the  end  of  twenty  four  hours  the 
milk  is  poured  through  a  fine  sieve  into  another  flask,  which  must  not  be 
more  than  four-fifths  full.  This  is  corked  and  allowed  to  stand,  being 
shaken  from  time  to  time.  At  the  end  of  twenty  four  hours  a  drink  is  ob- 
tained which  contains  but  little  COa  or  alcohol.  Usually  it  is  not  drunk 
until  the  second  day,  when,  upon  standing,  two  layers  are  formed,  the 
lower  milky,  translucent,  and  the  upper  containing  fine  flakes  of  casein. 
When  shaken  it  has  a  cream  like  consistence.  On  the  third  day  it  again 
becomes  thin  and  very  acid. 

"The  second  method  is  used  when  on**  has  a  good  kefir  of  two  or  three 
days  to  start  with.  Three  or  four  parts  of  fresh  cow's  milk  are  added  to  one 
part  of  this  and  poured  into  flasks  which  are  allowed  to  stand  for  forty- 
eight  hours  with  occasional  shaking  When  the  drink  is  i-eady  for  use'a 
portion  (one-fifth  to  one  third)  is  left  in  tho  flask  as  ferment  fora  fresh 
quantity  of  milk.  The  temperature  should  be  maintained  at  about  18°  O. ; 
but  at  the  commencement  a  higher  temperature  is  desirable.  The  korner 
snould  be  carefully  cleaned  from  time  to  time  and  broken  up  to  the  size  of 
j>«-as.  The  cleaned  korner  may  be  dried  upon  blotting  paper  in  the  sun  or 
in  the  vicinity  of  a  stove:  when  dried  in  the  air  they  retain  their  power  to 
germinate  for  a  long  time." 

Fermentation  of  urea.     The  alkaline  fermentation  of  urino  i< 


PRODUCTS   OF   VITAL   ACTIVITY.  137 

effected  by  various  microorganisms,  but  chiefly  by  the  Micrococcus 
ureae,  the  ferment  action  of  which  has  been  carefully  studied  by  Pas- 
teur, Duclaux,  and  others.  The  change  which  occurs  under  the 
action  of  the  living  ferment  was  determined  by  the  chemist  Dumas 
as  long  ago  as  1830,  but  it  remained  for  Pasteur  to  show  that  this 
change  depends  upon  the  presence  and  vital  activity  of  a  living 
microorganism. 

The  transformation  of  urea  into  carbonate  of  ammonia  is  shown 
by  the  following  formula  :  COH4ST0  +  2H2O  =  CO2  +  2NH9  + 
H,0  -  (NHJ.CO,. 

According  to  Van  Tiegheni,  Micrococcus  ureae  continues  to  grow 
in  a  liquid  containing  as  much  as  thirteen  per  cent  of  carbonate  of 
ammonia.  It  may  be  cultivated  in  an  artificial  solution  of  urea,  with 
the  addition  of  some  phosphates,  as  well  as  in  urine. 

The  Bacillus  ureae  of  Miquel  has  also  the  power  of  producing  the 
alkaline  fermentation  of  urine,  but  it  does  not  thrive  in  so  strong  a 
solution  of  carbonate  of  ammonia. 

A  different  micrococcus — Micrococcus  ureae  liquefaciens — nas  also 
been  studied  in  Fliigge's  laboratory  which  possesses  the  same  power. 
According  to  Musculus,  a  soluble  ferment  may  be  isolated  from  urine 
which  has  undergone  alkaline  fermentation,  which  changes  urea  into 
carbonate  of  ammonia.  He  obtained  it  from  urine  containing  con- 
siderable mucus,  in  a  case  of  catarrh  of  the  bladder.  But  Leube  has 
shown  that  cultures  of  Micrococcus  ureas  from  which  the  micrococ- 
cus was  removed  by  filtration  through  clay  do  not  induce  alkaline 
fermentation.  The  soluble  ferment  obtained  by  Musculus  must 
therefore  be  from  some  other  source. 

Miquel  has  given  special  attention  to  the  study  of  bacteria  which 
produce  alkaline  fermentation  in  urine,  and  in  addition  to  the  spe- 
cies above  mentioned  has  described  the  following  :  Urobacillus  Pas- 
teuri,  Urobacillus  Duclauxi,  Urobacillus  Freudenreichi,  Urobacillus 
Madcloxi,  Urobacillus  Schutzenbergi. 

Viscous  fermentation.  A  special  fermentation  which  occurs 
sometimes  in  wines,  and  in  the  juices  of  bulbous  roots  containing 
glucose,  and  in  milk,  is  produced  by  various  bacteria.  One  of  these 
is  a  micrococcus  which  has  been  described  by  Conn  under  the  name 
of  Micrococcus  lactis  viscosus.  The  fermented  juices  become  very 
viscous,  owing  to  the  formation  of  a  gum-like  product  resembling 
dextrin;  at  the  same  time  mannite  and  CO2  are  produced.  The 
gum-like  substance,  called  viscose  by  Bechamp,  is  soluble  in  cold 
water  and  is  precipitated  by  alcohol.  Guillebeau  (1892)  has  de- 
scribed a  micrococcus  and  a  bacillus  which  produce  viscous  fer- 
mentation in  milk — Micrococcus  Freudenreichi  and  Bacillus  Hessi. 


138  PRODUCTS   OF   VITAL   ACTIVITY. 

A  micrococcus  producing  viscous  fermentation  in  milk  has  also 
been  described  by  Schmidt-Miihlheim,  and  a  bacillus  by  Loffler. 
Bacillus  mesentericns  vnlgatus  also  produces  a  similar  change  in 
milk. 

Mai'fth  f/^.s-,  CH4,  is  produced  by  the  fermentation  of  cellulose, 
through  the  action  of  microorganisms  the  exact  characters  of  which 
have  not  yet  been  determined.  According  to  Tappeiner,  there  are 
two  different  fermentations  of  cellulose.  The  first  occurs  in  a  neu- 
tral one-per-cent  flesh  extract  solution  to  which  cotton  or  paper  pulp 
has  been  added.  The  gases  given  off  are  CO,  and  CH4  and  small 
quantities  of  H3S.  The  second  fermentation  occurs  when  an  alkaline 
solution  of  flesh  extract  containing  cellulose  in  suspension  is  used. 
The  gases  formed  are  CO,  and  H.  In  both  cases  small  quantities  of 
aldehyde,  isobutyric  acid,  and  acetic  acid  are  produced. 

Hydrosulphuric  acid,  H2S.  This  gas  is  produced  during  the 
growth  of  certain  bacteria.  The  conditions  governing  its  develop- 
ment have  been  studied  by  Holschewnikoff,  who  experimented  with 
two  species,  one  isolated  by  himself  and  one  by  Lindeiiborn,  named 
respectively  Bacterium  sulfureum  and  Proteus  sulfureus.  The  first- 
mentioned  bacterium,  when  inoculated  into  eggs,  produced  within 
three  or  four  days  an  abundant  quantity  of  H2S  ;  the  other  did  not. 
Upon  raw  albumin  both  species  produced  but  little,  and  011  the  yolk 
of  egg  a  considerable  amount  of  this  gas.  Upon  cooked  egg  the 
action  was  the  reverse.  In  peptone-bouillon  the  evolution  of  H2S 
was  abundant ;  in  the  absence  of  peptone,  very  slight. 

Putrefactive  fermentation.  The  putrefactive  decomposition 
of  albuminous  material  of  animal  and  vegetable  origin  is  effected 
by  a  great  variety  of  microorganisms  and  gives  rise  to  the  forma- 
tion of  a  great  variety  of  products,  some  of  which  are  volatile  and 
are  characterized  by  their  offensive  odors.  According  to  Flugge,  the 
first  change  which  occurs  consists  in  the  transformation  of  the  albu- 
mins into  peptone,  and  this  may  be  effected  by  a  large  number  of 
different  bacteria.  Among  the  products  of  putrefactive  fermenta- 
tion known  to  chemists  are  the  following  substances  :  Carbon  diox- 
ide, hydrogen,  nitrogen,  hydroBulphuric  acid  (H2S),  phosphoretted 
hydrogen  (PHJ,  methane,  formic  acid,  acetic  acid,  butyric  acid. 
valerianic  acid,  palmitic  acid,  crotonic  acid,  glycolic  acid,  oxalic 
acid,  succinic  acid,  propionic  acid,  lactic  acid,  amidostearic  acid, 
leucin,  ammonia,  ammonium  carbonate,  ammonium  sulphide,  tri- 
methylamine,  propylamine,  indol,  skatol,  tyrosin.neuridin,  cadaverin. 
putrescin,  cholin,  neurin,  peptotoxin.  and  various  other  volatile 
acids,  ptomaines,  etc. 

The  special  products  of  putrefaction  vary  according  to  the  nature 
of  the  material,  the  conditions  in  which  it  is  placed,  and  the  micro- 


PRODUCTS   OF   VITAL   ACTIVITY.  139 

organisms  present.  One  or  the  other  of  the  bacteria  concerned  will 
take  the  precedence  when  circumstances  favor  its  growth.  Thus  the 
aerobic  bacteria  cannot  grow  unless  the  putrefying  material  is  freely 
exposed  to  atmospheric  oxygen  ;  the  anaerobic  species  require  its 
exclusion.  Some  saprophytic  bacteria  grow  at  a  comparatively  low 
temperature,  others  take  the  precedence  when  the  temperature  is 
high  ;  some,  no  doubt,  thrive  only  in  presence  of  products  evolved 
by  other  species,  and  are  consequently  associated  with  and  depend- 
ent upon  these  species  ;  some  are  restrained  in  their  growth  sooner 
than  others  by  the  products  evolved  as  a  result  of  their  own  vital 
activity  or  that  of  associated  organisms  ;  some  grow  in  the  presence 
of  acids  and  give  rise  to  an  acid  fermentation  which  wholly  prevents 
the  development  of  other  species. 

At  the  outset  putrefaction  is  often  attended  with  the  presence 
of  several  species  of  micrococci  and  certain  large  bacilli,  which  are 
displaced  later  by  short  motile  bacteria  belonging  to  a  group  which 
includes  several  bacilli  formerly  described  under  the  common  name 
of  Bacterium  termo. 

The  malodorous  volatile  products  of  putrefaction  are  to  a  consid- 
erable extent  produced  by  anaerobic  species.  For  this  reason  these 
odors  are  more  pronounced  when  masses  of  albuminous  material 
undergo  putrefaction  in  situations  where  the  oxygen  of  the  air  has 
not  free  access  or  where  it  is  displaced  by  carbon  dioxide.  The 
body  of  a  dead  animal,  although  freely  exposed  to  the  air,  furnishes 
in  its  interior  a  suitable  nidus  for  these  anaerobic  gas-forming  spe- 
cies, and  they  may  give  rise  to  products  of  one  kind,  while  aerobic 
species  upon  the  surface  of  the  mass  induce  different  forms  of  putre- 
factive fermentation.  In  the  bodies  of  living  animals  these  anaero- 
bic microorganisms  are  constantly  present  in  the  intestine,  and  after 
death  they  quickly  invade  the  body  and  multiply  at  its  expense 
under  favorable  conditions  as  to  temperature.  The  surface  decom- 
position due  to  aerobic  bacteria  occurs  later  and  is  not  attended 
with  the  same  putrefactive  odors,  the  products  evolved  being  of  a 
simpler  chemical  composition — CO2,  HN3.  No  doubt  these  aerobic 
bacteria,  by  consuming  the  oxygen  and  forming  an  atmosphere  of 
carbon  dioxide,  help  to  make  the  conditions  favorable  for  the  con- 
tinued development  of  the  aiiaerobics  in  the  interior  of  the  organic  • 
mass  ;  at  the  same  time  they  find  a  suitable  pabulum  in  some  of  the 
more  complex  products  of  decomposition  occurring  in  the  absence 
of  oxygen.  The  gases  produced  in  the  interior  of  a  putrefying  mass 
are  mainly  CH4,  H2S,  and  H. 

Many  of  the  bacteria  of  putrefaction  are  facultative  aiiaerobics — 
that  is  to  say,  they  are  able  to  multiply  either  in  the  presence  of  oxy- 
gen or  in  its  absence.  The  products  evolved  by  these  differ,  no 


140  PRODUCTS   OF   VITAL   ACTIVITY. 

doubt,  arc, .nling  to  whether  they  are  or  are  not  supplied  with  atmos- 
pheric oxygen. 

The  anaerobic  bacteria  concerned  in  putrefaction  have  as  yet 
received  comparatively  little  attention.  Among  the  aerobics  and 
facultative  anaerobics  the  following  are  best  known:  Micrococcus 
foetidus,  Bacillus  saprogenes  I.,  II.,  and  III.,  Bacillus  coprogenes- 
foetidus,  Bacillus  putrificus  coli,  Proteus  vulgaris,  Proteus  Zenkeri, 
Proteus  mirabilis,  Bacillus  pyogeues  foetidus,  Bacillus  fluorescens 
liquefaciens,  Bacillus  pyocyaneus,  Bacillus  coli  communis,  Bacillus 
janthinus. 

Sithihh'  /-V/ -im-iifti. — Several  species  of  bacteria  produce  soluble 
ferments  capable  of  changing  starch  into  maltose,  dextrin,  etc. 
Hueppe  has  shown  that  the  lactic-acid  bacillus  produces  a  diastase, 
and  Miller  obtained  1'nnn  the  human  intestine  a  species  which  dis- 
-olves  starch.  Marcano,  by  filtering  cultures  of  species  capable  of 
this  ferment  action  through  porcelain.  \vas  able  to  show  that  the 
effect  is  due  to  a  soluble  ferment,  which  must  have  been  produced 
by  the  vital  activity  of  the  living  microorganisms.  Wortmann  also 
obtained  a  diastase  from  culture  liquids  which  was  precipitated  by 
alcohol  ;ind  again  dissolved  in  water:  in  slightly  acid  solutions  it 
promptly  converted  starch  into  glucose.  This  is  said  to  be  produced 
in  culture  liquids  only  when  these  do  not  contain  albumin.  In  the 
presence  of  all  mini  n  a  peptonizing  ferment  was  formed;  in  its  ab- 

••e.  a  diastase  by  which  starch  was  dissolved  to  serve  as  pabulum 
for  the  bacteria  present.  These  experiments  were  not  made  with 
pure  cultures,  and  more  exact  researches  in  this  direction  are  de- 
niable. 

A  peptonizing  ferment  for  gelatin  is  produced  by  a  considerable 
number  of  bacteria,  as  stated  under  the  heading  "Liquefaction  of 
Gelatin."  The  jellified  albumin  in  cultures  in  blood  serum  is  also 
liquefied  by  a  peptoni/in-  ferment  prod  need  by  certain  species  of  ba.-- 

>'•  authors  also  speak  of  a  soluble  ferment  capable  of  inverting 

cane  sugar  or   milk   >n-ar.      According  to  Hueppe.   such   a    ferment 

iced  by  the  Bacillus  acidi  lactici.      A  soluble  ferment  for  cel- 

hlkMH  is  Mippo^-d   by  Flttgge  to  be  produced   by  several  species— 

•mOOg  Others  by   Bacillus  butyricus  and  by  Vibrio  ruguhi. 

Sev.-ral  bacilli  produce  a  soluble  ferm.'iit  capable  of  coa-'ulatiiiLC 
tin-  'MM»in  of  milk. 

l:""</  \ftrates,  «„<!   \i'tnn<;,fi<>n.—The  researches  of 

Gayon.  hupettit.  and  others  show  that  certain  bacteria  are  able  to 
reduce  nitrates  with  liberation  qf  ammonia  and  free  nitrogen.  This 
is  effected  in  the  absence  of  oxygen  by  anaerobic  bacteria,  and. 


PRODUCTS   OF   VITAL   ACTIVITY.  141 

among  others,  lay  Bacillus  butyricus.  Certain  aerobic  bacteria  also 
accomplish  the  same  result.  Thus  Herseus  obtained  two  species 
from  water  which  reduced  nitrates  in  a  very  decided  manner.  On 
the  other  hand,  a  number  of  species  are  known  to  oxidize  ammonia, 
producing  nitric  acid.  Schlosing  and  Miinz,  as  a  result  of  numerous 
experiments,  arrived  at  the  conclusion  that  in  the  soil  nitrification  is 
effected  by  a  single  species.  But  it  is  doubtful  whether  they  worked 
with  pure  cultures,  and  more  recent  researches  show  that  several, 
and  probably  many,  different  bacteria  possess  this  power.  Accord- 
ing to  Heraaus,  the  following  species,  tested  by  him,  oxidize  am- 
monia :  Bacillus  prodigiosus,  the  cheese  spirillum  of  Deneke,  the 
Finkler-Prior  spirillum,  the  typhoid  bacillus,  the  anthrax  bacillus, 
the  staphylococci  of  pus.  The  oxidation  does  not  always  go  to  the 
point  of  forming  nitrates,  but  nitrites  may  be  formed  in  the  soil 
(Duclaux).  Warrington  states  that  certain  bacteria  which  formed 
nitrates  in  a  suitable  culture  medium  produced  only  nitrites  when, 
after  an  interval  of  four  or  five  months,  some  of  the  culture  was 
transferred  to  a  solution  containing  muriate  of  ammonia.  The  same 
author  states  that  the  process  of  nitrification  occurs  only  in  the 
dark. 

The  researches  of  Winogradsky,  of  the  Franklands,  and  of  Jor- 
dan show  that  the  failure  of  earlier  investigators  to  obtain  the  nitri- 
fying bacteria  from  the  soil  in  pure  cultures  was  due  to  the  fact  that 
these  bacteria  do  not  grow  in  the  usual  culture  media.  By  the  use 
of  certain  saline  solutions  the  authors  named  have  succeeded  in  iso- 
lating nitrifying  bacteria  in  pure  cultures,  or  nearly  so.  It  is  still 
uncertain  whether  these  investigators  have  obtained  the  same  bac- 
teria, but  the  microorganisms  described  by  them,  and  obtained  from 
widely  distant  sources,  are  similar  in  their  morphological  and  bio- 
logical characters,  and  at  least  belong  to  the  same  group.  In  a  com- 
munication made  in  1891  Winogradsky  arrives  at  the  conclusion 
that  the  ferments  which  cause  the  oxidation  of  ammonia  and  pro- 
duction of  nitrites  are  not  capable  of  producing  nitrates,  but  that 
other  microorganisms  are  concerned  in  the  oxidation  of  nitrites. 
In  sterilized  soil  to  which  a  pure  culture  of  his  nitromonas  was 
added  nitrites  only  were  produced,  and  the  presence  of  various 
microorganisms  common  in  the  soil  did  not  result  in  the  forma- 
tion of  nitrates  so  long  as  the  specific  ferment  was  absent  to  which 
this  second  oxidation  is  ascribed  (nitrifying  bacillus  of  Winograd- 
sky). 

Phosphorescence. — Several  different  bacteria  have  been  studied 
which,  in  pure  cultures,  give  rise  to  phosphorescence  in  the  medi- 
um in  which  they  are  cultivated.  In  gelatin  cultures  the  light 
is  sufficient  in  some  instances  to  enable  one  to  tell  the  time  by  a 


11-.'  PROMPTS   OF   VITAL    ACTIVITY. 

watch  in  a  |*?rfectly  dark  room,  and  such  cultures  have  even  been 
photographed  by  their  own  light. 

The  phosphorescence  is  influenced  by  changes  in  the  culture 
medium  and  by  conditions  of  temperature,  but  we  have  no  exact 
knowledge  of  the  mode  of  its  production.  The  Bacillus  phosphores- 
<  ins  from  sea  water  in  the  vicinity  of  the  West  Indies  gives  the 
must  striking  results,  especially  when  planted  upon  the  surf  ace  of 
cooked  fish  and  placed  in  an  incubating  oven  at  30°  C.  Two  other 
species  have  been  studied  by  Fischer — one  obtained  from  the  water 
of  the-  harl>or  at  Kiel,  and  the  other  a  widely  distributed  species 
•  •ailed  by  Fischer  Bacterium  phosphorescens.  Katz  (1801)  has  de- 
-« -rilied  several  species  obtained  by  him  from  sea  water  and  from 
phosphorescent  fish  in  the  markets  at  Sydney,  New  South  Wales — 
Bacillus  smaragdino-phosphorescens,  Bacillus  argenteo-phosphores- 
cens,  Bacillus  cyaneo-phosphorescens,  Bacillus  argenteo-phosphores- 
cens  liquefaciens. 


V. 
PTOMAINES    AND    TOXALBUMINS. 

VARIOUS  basic  substances  containing  nitrogen,  and  in  chemical 
constitution  resembling  the  vegetable  alkaloids,  have  been  isolated 
by  chemists  from  putrefying  material  and  from  cultures  of  the  bac- 
teria concerned  in  putrefaction,  and  also  from  certain  pathogenic 
species.  Some  of  these  ptomaines  are  non-toxic,  and  others  are 
very  poisonous  in  minute  doses  (toxines).  The  toxic  substances 
sometimes  developed  in  milk,  cheese,  sausage,  etc.,  are  also  of  this 
nature,  and  are  doubtless  produced  by  the  action  of  microorganisms. 
The  pathogenic  power  of  the  bacteria  which  cause  various  infectious 
diseases  in  man  and  the  lower  animals  has  also  been  shown  to  result 
from  the  production  of  toxic  ptomaines  or  of  toxalbumins.  Selmi  first 
gave  the  name  ptomaines  to  cadaveric  alkaloids  isolated  by  him,  and 
Panum  subsequently  called  attention  to  the  fact  that  poisonous  basic 
substances  of  this  class  are  contained  in  putrefying  material.  Ex- 
tended researches  with  reference  to  the  ptomaines  have  since  been 
made  by  numerous  chemists,  the  most  important  being  those  of  Berg- 
mann,  Schmiedeberg,  Zuelzer  and  Sonnenschein,  Hager,  Otto,  Sel- 
mi, Brieger,  Gautier  and  Etard,  and  Vaughan. 

For  a  full  account  of  the  history  and  chemical  composition  of  the 
ptomaines  the  reader  is  referred  to  the  valuable  work  of  Vaughan 
and  Novy  ("  Ptomaines  and  Leucomaines,"  Philadelphia,  1891).  In 
the  present  volume  we  shall  give  a  brief  account  only  of  some  of  the 
most  important. 

NON-TOXIC   PTOMAINES. 

Neuridin,  C5HJ4N2. — This  is  one  of  the  most  common  of  the  al- 
kaloids of  putrefaction  and  was  isolated  by  Brieger  in  1884.  It  is 
obtained  most  abundantly  from  tissues  containing  gelatin.  Very 
soluble  in  water,  but  insoluble  in  ether  and  absolute  alcohol.  Has  a 
disagreeable  odor. 

Cadaverin,  C6HMN3. — Isomeric  with  iieuridin  ;  has  a  very  dis- 
agreeable odor  ;  forms  a  thick,  transparent,  syrupy  liquid  ;  is  vola- 
tile, and  can  be  distilled  with  steam  without  undergoing  decomposi- 
tion. When  exposed  to  the  air  the  base  absorbs  carbon  dioxide  and 


144  PTOMAINKS   AND   TOXALBl'MINS. 

form>  a  crystalline  mass.  Is  produced  in  cultures  of  the  cholera 
>j.ii  ilium  and  of  the  spirillum  of  Finkler  and  Prior  which  have  been 
kept  for  a  month  or  more  at  37°  C. 

Putretcin,  C4H1SN,.— A  base  resembling  cadaverin  and  com- 
1 1 1<  mly  associated  with  it.  Obtained  by  Brieger  from  various  sources, 
most  abundantly  from  substances  containing  gelatin  and  in  the 
in-  »re  advanced  stages  of  putrefaction.  It  is  obtained  in  the  form  of 
a  hydrate,  which  is  a  transparent  liquid  having  a  boiling  point  of 
a  bou  t  1  :'».")0.  With  acids  it  forms  crystalline  salts. 

X'i/n'in.  Ct A ,.N2.—  Resembles  cadaverin  and  is  commonly  as- 
B  „  Mated  with  it  in  putrefying  material.  Isolated  by  Brieger. 

Met  Ill/In  mine,  CHS.NH,.— Obtained  by  Brieger  from  putrefying 
ti-li  and  from  old  cultures  of  the  cholera  spirillum. 

Dinn'f  In/la  mine,  (CH,),.NH. — Obtained  by  Brieger  from  putre- 
fying p -la tin  and  by  Bocklisch  from  decomposing  fish. 

Trt'im-f  hi/him  inc.  (CH,)SN. — Obtained  from  various  sources,  and 
l»y  Brieger  from  cultures  of  the  cholera  spirillum  and  of  the  strepto- 

•ns  «»r  pu-. 

TOXIC  PTOMAINES. 

\ ••'iriii,  CftH,,NO. —First  obtained  by  Liebreich  in  1865  as  a 
•  I.  <  ,.nijK>sition  product  of  protagon  from  the  brain.  Obtained  by 
Brieger  from  putrefying  muscular  tissue.  When  crystallized  from 
an  aqueous  solution  it  forms  five-  or  six-sided  plates  ;  from  an  alco- 
holic solution  it  crystallizes  in  the  form  of  needles  (Liebreich).  This 
-e,  is  toxic  in  small  doses.  In  frogs  the  injection  of  a  few  milli- 

1 1 lines  produces  paralysis  of  the  extremities.  Respiration  is  first 
a  r  posted  and  the  heart  stops  in  diastole.  Atropine  appears  to  be  a 
physiological  antidote  to  the  toxic  effects  of  neurin.  In  rabbits  it 
produces  profuse  salivation.  The  pupil  is  contracted  by  the  direct 
application  of  a  concentrated  solution. 

<'!„,/ in..  C.H..NO,.— First  obtained  from  hog's  bile  by  Strecker 
in  1862.  Has  been  obtained  by  Brieger  from  various  sources,  in- 
« •hiding  i Mil tn ITS  of  the  cholera  spirillum.  It  is  also  found  widely 
di-trihuted  in  the  vegetable  kingdom.  Maybe  prepared  from  the 
s  oik  of  eggs  by  the  method  of  Diakonow.  Cholin  is  obtained  in  the 
t'onn  of  a  syrupy,  alkaline  liquid  which  combines  with  acids  to  form 
deliquescent  salt-.  At  first  this  base  was  not  supposed  to  have  toxic 
properties.  l»ut  more  recent  researches  have  shown  that  in  compara- 
tively large  doses  it  produces  symptoms  resembling  those  caused  by 
minute  doses  of  m-nrin. 

Mu8carin,  C,HUNO,.—  This  toxic  principle  of  poisonous  mush- 

•  i ns  has  al so  h,  M  n  <  >btained  by  Brieger  f  r<  >m  pu t  ref ying  fish.  It  may 
be  produ.-.-d  artiti.-ially  hy  tin-  oxidation  of  oholin.  In  small  doses 
it  kilU  rahhit<  and  frogs.  In  the  rabbit  it  produces  lacrymation  and 


PTOMAINES   AND   TOXALBUMINS.  145 

salivation,  the  pupil  is  contracted,  and  the  animal  dies  in  convul- 
sions. Frogs  are  completely  paralyzed  by  the  action  of  muscarin 
and  die  with  arrest  of  the  heart's  action  in  diastole. 

Peptotoxin. — The  exact  composition  of  this  ptomaine  has  not 
been  determined.  Brieger  obtained  it  during  the  early  putrefac- 
tion of  proteid  substances  and  also  from  the  artificial  digestion  of 
fibrin.  It  is  very  poisonous  for  frogs,  which  become  paralyzed  and 
die  within  fifteen  or  twenty  minutes  after  the  subcutaneous  injection 
of  a  few  drops  of  a  dilute  solution.  Rabbits  also  are  killed  by  doses 
of  half  a  gramme  to  a  gramme,  the  symptoms  being  paralysis  of  the 
posterior  extremities  and  stupor.  Peptotoxin  is  soluble  in  water, 
but  insoluble  in  ether  or  chloroform.  It  is  not  destroyed  by  boiling. 

Tyrotoxicon. — First  obtained  by  Vaughan  in  poisonous  cheese, 
and  subsequently  by  the  same  chemist  and  others  in  poisonous  milk 
and  ice  cream.  Chemically  tyrotoxicon  is  very  unstable.  It  is  de- 
composed when  heated  with  water  to  90°  C.  It  is  insoluble  in  ether. 
From  sixteen  kilogrammes  of  poisonous  cheese  Yaughan  obtained 
0. 5  gramme  of  the  poison.  The  symptoms  produced  in  man  by  eat- 
ing cheese  or  milk  containing  tyrotoxicon  are  vertigo,  nausea,  vomit- 
ing, and  severe  rigors,  with  pain  in  the  epigastrium,  cramps  in  the 
legs,  griping  pain  in  the  bowels  attended  with  purging,  numbness 
and  a  pricking  sensation  in  the  limbs,  and  great  prostration. 

Methyl-guanidin,  C3H7N3. — Obtained  by  Brieger  from  putrefy- 
ing horseflesh  which  had  been  kept  at  a  low  temperature  for  several 
months.  This  base  was  previously  known  to  chemists,  having  been 
obtained  by  the  oxidation  of  creatin.  By  Bocklisch  it  has  been  ob- 
tained from  impure  cultures  of  the  Finkler-Prior  spirillum  which 
had  been  kept  for  about  a  month.  It  is  obtained  as  a  colorless  mass 
having  an  alkaline  reaction,  and  which  is  quite  deliquescent.  Brie- 
ger gives  the  following  account  of  the  toxic  action  as  tested  on 
guinea-pigs  in  a  dose  of  0. 2  gramme  :  The  respiration  increases  in 
rapidity,  the  pupils  dilate  to  the  extreme  limit,  the  animal  has  copi- 
ous discharges  of  urine  and  faeces,  the  extremities  become  paralyzed, 
and  at  the  end  of  about  twenty  minutes  death  occurs  in  convulsions. 

Mytilotoxin. — Obtained  by  Brieger  from  poisonous  mussels. 
The  toxic  action  resembles  that  of  curare. 

Typhotoxtn,  C7H17NO2. — Obtained  by  Brieger  from  bouillon 
cultures  of  the  typhoid  bacillus  which  had  been  kept  for  a  week  or 
more  at  a  temperature  of  about  37.5°  C.  In  mice  and  guinea-pigs 
this  base  produces  salivation,  rapid  respiration,  dilatation  of  the 
pupils,  diarrhoea,  and  death  in  from  twenty-four  to  forty-eight  hours. 
It  is  believed  by  Brieger  that  the  specific  action  of  the  typhoid  bacil- 
lus is  due  to  the  production  of  this  ptomaine. 

A  base  which  is  isomeric  with  typhotoxin  has  been  obtained  by 
10 


PTOMAINES  AND   TOXALBUMINS. 

Brieger  from  putrefying  horseflesh  which  was  kept  at  a  low  tempe- 
rature for  several  months.  Unlike  it,  however,  the  free  base  has 
an  acid  reaction,  while  typhotoxin  is  strongly  alkaline.  It  differs  also 
in  its  physiological  action,  being  more  toxic  and  producing  convul- 
sions ;  the  heart  is  arrested  in  diastole.  Typhotoxin,  on  the  other 
hand,  does  not  induce  convulsions  and  the  heart  is  arrested  in  systole. 

Tetanin,  C,,H,0N,O4.—  Obtained  by  Brieger  from  impure  cul- 
tures of  the  tetanus  bacillus  cultivated  in  bouillon  in  an  atmosphere 
of  hydrogen.  (The  tetanus  bacillus  is  a  strict  anaerobic.)  Obtained 
subsequently  by  the  same  chemist  from  the  amputated  arm  of  a  pa- 
tient with  tetanus.  This  base  has  been  obtained,  by  crystallization 
from  hot  alcohol,  in  clear  yellow  plates  which  are  not  very  soluble  in 
water.  The  hydrochloride  is  a  deliquescent  salt  which  dissolves 
readily  in  alcohol.  When  injected  into  guinea-pigs  or  mice  in  rather 
large  doses,  tetanin  first  causes  the  animal  to  fall  into  a  lethargic 
condition,  followed  by  increased  rapidity  of  respiration  and  tetanic 
convulsions.  In  guinea-pigs  opisthotonos  is  induced,  together  with 
the  characteristic  tetanic  convulsions  as  seen  in  animals  suffering  from 
tetanus.  Three  other  toxic  bases  have  been  obtained  by  Brieger 
from  cultures  of  the  tetanus  bacillus,  which  cause  similar  symptoms. 
One — tetanotoxin — is  given  by  Brieger  the  formula  C5HMN.  A 
second  base,  the  composition  of  which  has  not  been  determined,  is 
called  spasmotoxin. 

Cholera  Ptomaines. — Brieger  has  obtained  from  pure  cultures 
of  the  cholera  spirillum  several  of  the  toxic  ptomaines  heretofore  re- 
ferred to — cadaverin,  putrescin,  cholin,  methyl-guanidin.  In  addi- 
tion to  these  he  found  two  toxic  substances  which  appear  to  be  pe- 
culiar products  of  this  microorganism.  One  induces  cramps  and 
muscular  tremors  in  small  animals,  the  other  diarrhoea  and  symp- 
toms of  collapse. 

Toxalbumins. — Researches  by  Brieger  and  Frankel  (1890)  show 
that  very  toxic  substances  of  a  different  nature  are  present  in  cultures 
of  some  of  the  pathogenic  bacteria ;  these  have  been  designated  by  the 
authors  named  "toxalburnins." 

Roux  and  Yersin  had  previously  shown  that  filtered  cultures  of  the 
diphtheria  bacillus  contain  a  toxic  substance  which  produces  paralysis 
ami  death  in  ^ninea-pi-s  and  rabbits.  This  substance  lias  now  been 
obtained  in  a  pure  state  and  its  toxic  action  tested  by  the  authors 
ti''st  ii.-nm-d.  It  is  destroyed  by  a  temperature  of  On  ('.,  but  remains 
in  an  active  condit  i«  .n  in  cultures  which  have  been  sterilized  by  seve- 
ral hours'  exposure  to  a  temperature  of  50°,  or  in  those  which  have 
IMM-II  passed  thro.,-!,  a  clay  filter.  It  is  not  volatile,  and  differs  essen- 
li;lll.v  fr"m  tll(1  i't"inainosand  also  from  the  soluble  ferments.  It 
was  obtained  as  a  snow-white,  amorphous  mass  which  was  ex- 


PTOMAINES    AND    TOXALBUMINS.  14? 

tremely  toxic  in  its  action  upon  small  animals.  When  injected  into 
guinea-pigs  in  the  proportion  of  two  and  one-half  milligrammes  to 
one  kilogramme  of  body  weight,  it  caused  death  after  a  considerable 
interval  of  time  (from  a  few  days  to  several  weeks),  during  which 
the  animal  became  emaciated  and  spreading  abscesses  and  necrosis 
of  the  tissues  occurred  at  the  point  of  injection.  This  toxalbumin 
was  obtained  in  a  pure  state  by  repeated  precipitation  from  an  aque- 
ous solution  by  means  of  alcohol.  It  is  produced  most  abundantly 
in  cultures  containing  albumin,  and  old  cultures  are  more  toxic  than 
recent  ones.  Chemical  analysis  gave  the  following  result  :  C  45,  35, 
H  7,  13,  N  16,  33,  S  1,  39, 'O  29,  80.  The  authors  remark,  however, 
that  the  chemical  characters  have  not  yet  been  fully  determined. 

The  same  chemists  have  obtained  toxic  substances  of  a  similar 
nature  from  cultures  of  the  bacillus  of  typhoid  fever,  of  the  tetanus 
bacillus,  of  the  Staphylococcus  aureus,  and  of  the  cholera  spirillum. 
Hankin  had  previously  obtained  a  toxic  "albumose"  from  cultures 
of  the  anthrax  bacillus  by  precipitation  with  alcohol,  drying,  solu- 
tion in  water,  and  filtration  through  porcelain ;  and  Christmas  had 
obtained  an  albuminous  substance  from  cultures  of  Staphylococcus 
aureus  which  produced  pus  formation  when  injected  beneath  the 
skin  of  rabbits  or  into  the  anterior  chamber  of  the  eye. 

According  to  Brieger  and  Frankel,  these  toxalbumins  are  divided 
into  two  principal  groups,  one  of  which  is  characterized  by  solubility 
in  water,  as  in  that  produced  by  the  diphtheria  bacillus  ;  and  one  in 
which  the  albumin  is  insoluble  or  but  slightly  soluble,  as  is  the  case 
with  those  obtained  from  cultures  of  the  typhoid  bacillus,  the  cholera 
spirillum,  and  the  Staphylococcus  aureus. 

The  toxalbumin  from  cholera  cultures,  obtained  as  pure  as  pos- 
sible and  suspended  in  water,  when  injected  under  the  skin  of  a 
guinea-pig,  caused  its  death  in  two  or  three  days.  It  was  not,  how- 
ever, toxic  for  rabbits,  even  when  injected  in  considerable  quantity. 

On  the  contrary,  the  toxalbumin  of  the  typhoid  bacillus,  which  is 
dissolved  with  difficulty  in  water,  was  more  poisonous  for  rabbits 
than  for  guinea-pigs.  When  injected  subcutaneously  into  rabbits 
death  usually  occurred  in  eight  to  ten  days.  No  notable  pathologi- 
cal changes  were  observed  at  the  autopsy. 

The  toxalbumin  of  Staphylococcus  aureus  killed  rabbits  and 
guinea-pigs  within  a  few  days,  and  in  some  cases  at  the  end  of 
twenty-four  hours.  The  post-mortem  appearances  were  necrosis  or 
purulent  breaking  down  of  the  tissues  at  the  point  of  injection,  with 
swelling  and  redness  of  the  surrounding  tissues  and  general  inflam- 
matory appearances.  The  toxalbumin  of  anthrax  cultures  resembles 
that  of  the  diphtheria  bacillus  in  being  soluble  in  water.  It  was 
obtained  by  Brieger  from  the  organs  of  animals  recently  dead  from 


148  PTOMAINES   AND   TOXALBUMINS. 

anthrax.  In  a  dry  condition  it  has  a  grayish-white  color  and  gives 
the  reactions  of  albumins. 

The  toxalbumin  of  the  tetanus  bacillus  is  also  soluble  in  water. 
It  is  best  obtained  in  bouillon  cultures  containing  glucose. 

G.  and  F.  Klemperer  (1891)  have  announced  their  success  in 
obtaining  a  toxalbumin  from  cultures  of  Micrococcus  pneumonias 
crouposse  ('diplococcus  pneumonia*') ;  this  they  propose  to  call  pneu- 
motoxin. 

Koch's  "  Tuberculin."— This,  is  a  glycerin  extract  of  the  toxic 
substances  present  in  cultures  of  the  tubercle  bacillus.  Crude  tu- 
berculin is  obtained  from  liquid  cultures  made  in  veal  broth  to  which 
one  per  cent  of  peptone  and  four  to  five  per  cent  of  glycerin  have 
been  added.  This  culture  liquid  is  placed  in  flasks  and  inoculated 
upon  the  surface  with  small  masses  from  a  pure  culture  of  the  tu- 
bercle bacillus.  A  tolerably  thick  and  dry  white  layer  is  developed, 
which  after  a  time  covers  the  entire  surface.  At  the  end  of  six  to 
eight  weeks  development  ceases  and  the  culture  liquid  is  evaporated 
over  a  water  bath  to  one-tenth  its  volume  ;  this,  after  being  filtered, 
constitutes  the  crude  tuberculin.  By  precipitation  with  sixty-per- 
cent alcohol  Koch  has  obtained  from  this  a  white  precipitate  which 
has  the  active  properties  of  the  glycerin  extract.  This  is  soluble  in 
water  and  in  glycerin,  and  has  the  chemical  reactions  of  an  albumi- 
nous body. 

Zuelzer  has  (1801)  reported  his  success  in  isolating  atoxic  sub- 
stance from  tubercle  cultures.  The  contents  of  tubes  containing 
pure  cultures  of  the  bacillus  are  first  treated  with  hot  water 
acidulated  with  hydrochloric  acid.  This  solution  is  filtered,  evapo- 
rated, and  then  several  times  precipitated  with  platinum  chloride. 
The  double  salt  formed  is  decomposed  by  hydrosulphuric  acid, 
after  which  the  liquid  is  filtered  and  evaporated  to  dryness.  A 
white,  crystalline  salt  is  thus  obtained  which  is  soluble  in  hot  water. 
This  salt  was  toxic  for  rabbits  and  guinea-pigs  in  doses  of  from  one 
to  three  centigrammes.  Death  usually  occurred  in  from  two  to  four 
days.  In  guinea-pigs  one  centigramme  injected  subcutaneously 
caused,  within  a  few  minutes,  a  greatly  increased  frequency  of  respi- 
ration, an  elevation  of  temperature,  and  protrusion  of  the  eyeballs. 

Malle'in. — Kalwing,  Preusse,  and  Pearson  have  obtained  from 
cultures  of  the  glanders  bacillus  a  "lymph"  which  somewhat  re- 
sembles the  crude  tuberculin  of  Koch.  This  was  obtained  by 

1'ivussc  l.y  tivjitin^-  <>1«1  j»ot;it<  >  cult  mvs  of  thr  glanders  bacillus  with 
glycerin  and  water.  The  extract  was  filtered  several  times  and  then 
sterilized  in  a  steam  stn-ili/er.  This  lymph  injected  into  horses  in- 
fected with  glanders  gives  rise  to  a  very  decided  elevation  of  tempe- 
ratuiv.  \\  liilr  iu  horses  free  from  this  disease  no  such  result  follows. 


VI. 

INFLUENCE  OF  PHYSICAL  AGENTS. 

Heat. — We  have  already  seen  (Section  II. ,  Part  Second)  that  the 
temperature  favorable  for  the  growth  of  most  bacteria  is  between  20° 
and  40°  C. ;  that  some  species  are  able  to  multiply  at  the  freezing  tem- 
perature, and  others  at  as  high  a  temperature  as  60°  to  70°  C. ;  that, 
as  a  rule,  the  parasitic  species  require  a  temperature  of  35°  to  40°; 
and  that  low  temperatures  do  not  kill  bacteria. 

Frisch  (1877)  exposed  various  cultures  to  a  temperature  of  —87°  C., 
which  he  obtained  by  the  evaporation  of  liquid  CO2,  and  found  that 
micrococci  and  bacilli,  after  exposure  to  such  a  temperature,  multi- 
plied abundantly  when  again  placed  in  favorable  conditions.  Prud- 
den  has  also  made  extended  experiments  upon  the  influence  of 
freezing.  He  found  that  while  certain  species  resisted  the  freezing 
temperature  for  a  long  time,  others  failed  to  grow.  Thus  Bacillus 
prodigiosus  did  not  grow  after  being  frozen  for  fifty-one  days  ;  Pro- 
teus vulgaris  was  killed  in  the  same  time,  and  a  slender,  liquefying 
bacillus  obtained  from  Croton  aqueduct  water  was  killed  in  seven 
days.  Staphylococcus  pyogenes  aureus  withstood  freezing  for  sixty- 
six  days,  a  fluorescent  bacillus  from  Hudson  River  ice  for  seventy- 
seven  days,  and  the  bacillus  of  typhoid  fever  for  one  hundred  and 
three  days.  Cultures  made  at  intervals  showed,  however,  a  dimi- 
nution in  the  number  of  bacteria.  A  similar  diminution  would  per- 
haps have  occurred  in  old  cultures  in  which  the  pabulum  for  growth 
was  exhausted,  independently  of  freezing  ;  for  bacteria,  like  higher 
plants,  die  in  time — which  varies  for  different  species — as  a  result  of 
degenerative  changes  in  the  living  protoplasm  of  the  cells,  and  con- 
tinued vitality  in  a  culture  depends  upon  continued  reproduction. 

Repeated  freezing  and  thawing  was  found  by  Prudden  to  be 
more  fatal  to  the  typhoid  bacillus  than  continuous  freezing.  Cul- 
tures were  sterilized  by  being  thawed  out  at  intervals  of  three  days 
and  again  ref rozen,  after  repeating  the  operation  five  times. 

Cadeac  and  Malet  kept  portions  of  a  tuberculous  lung  in  a  frozen 
condition  for  four  months,  and  found  that  at  the  end  of  this  time 
tuberculosis  was  still  produced  in  guinea-pigs  by  injecting  a  small 
quantity  of  this  material. 


]50  INFLUENCE  OF  PHYSICAL  AGENTS. 

In  considering  the  influence  of  high  temperatures  we  must  take 
account  of  the  very  great  difference  in  the  resisting  power  of  the 
vegetative  cells  and  the  reproductive  elements  known  as  spores,  also 
of  the  fact  as  to  whether  dry  or  moist  heat  is  used  and  the  time  of 

exposure. 

Dry  Heat.— When  microorganisms  in  a  desiccated  condition  are 
exposed  to  the  action  of  heated  dry  air,  the  temperature  required  for 
their  destruction  is  much  above  that  required  when  they  are  in  a 
moist  condition  or  when  they  are  exposed  to  the  action  of  hot  water 
or  steam.  This  was  thoroughly  demonstrated  by  the  experiments  of 
Koch  and  Wolffhugel  (1881).  A  large  number  of  pathogenic  and 
non-pathogenic  species  were  tested,  with  the  following  general  result : 
A  temperature  of  78°  to  123°  C.  maintained  for  an  hour  and  a  half 
(over  100°  for  an  hour)  failed  to  kill  various  non-pathogenic  bacteria, 
but  was  fatal  to  the  bacillus  of  mouse  septicaemia  and  that  of  rabbit 
septicaemia.  To  insure  the  destruction  of  all  the  species  tested,  in 
the  absence  of  spores,  a  temperature  of  120°  to  128°  C.,  maintained 
for  an  hour  and  a  half,  was  required. 

The  spores  of  Bacillus  anthracis  and  of  Bacillus  subtilis  resisted 
tins  temperature  and  required  to  insure  their  destruction  a  tempera- 
ture of  140°  C!  maintained  for  three  hours.  This  temperature  was 
found  to  injure  most  objects  requiring  disinfection,  such  as  clothing 
and  bedding.  But  the  lower  temperature  which  destroys  micro- 

;;inisms  in  the  absence  of  spores  (120°  C.  =  248°  F.)  can  be  used 
for  disinfecting  articles  soiled  with  the  discharges  of  patients  with 
cholera,  typhoid  fever,  or  diphtheria,  as  the  specific  germs  of  these 
« 1 1  Ceases  do  not  form  spores.  It  is  probable  also  that  it  may  be  safely 
use<l  to  disinfect  the  clothing  of  small-pox  patients,  for  we  have  ex- 
perimental evidence  that  a  lower  temperature  destroys  the  virulence 
iccine  virus  (90°-95°  C. — Baxter). 

I  ii  practical  disinfection  by  means  of  dry  heat  it  will  be  necessary 
to  remember  that  it  has  but  little  penetrating  power.  In  the  experi- 
ments of  Koch  and  Wolffhugel  it  was  found  that  registering  ther- 
mometers placed  in  the  interior  of  folded  blankets  and  packages  of 
\.irions  kinds  did  not  shmv  a  temperature  capable  of  killing  bacteria 
after  three  hours*  exposure  in  a  hot-air  oven  at  133°  C.  and  above. 

Moist  Heat.— The  thermal  death-point  of  bacteria,  in  the  ab- 
•SHO8  Of  Spores,  |g  Comparatively  lo\v  when  they  are  exposed  to  moist 
beat.  The  results  of  the  writer's  experiments  are  given  below : 

In  my  temperature  experiments  I  have  taken  great  pains  to  insure  the 
exposure  of  the  test  organisms  to  a  uniform  temperature,  and  have  adopted 
tM.  minutes  as  the  standard  time  of  exposure.  The  method  employed 
throughout  lias  been  as  follows:  From  glass  tubing  having  a  diameter  of 
about  three-sixteenths  of  an  inch  1  draw  out  in  the  flameof  a  Bunsen  burner 
a  nanMr  Of  capillary  tubes,  with  an  •  •xpandrd  extremity  which  serves  as 


INFLUENCE    OF   PHYSICAL   AGENTS. 


151 


an  air  chamber.  A  little  material  from  a  pure  culture  of  the  test  organ- 
ism is  drawn  into  each  of  these  capillary  tubes  by  immersing  the  open 
extremity  in  the  culture,  after  having-  gently  heated  the  expanded  end.  The 
end  of  the  tube  is  then  hermetically  sealed  by  heat.  These  tubes  are  im- 
mersed in  a  water  bath  maintained  at  the  desired  temperature  for  the  stan- 
dard time.  The  bath  is  kept  at  a  uniform  temperature  by  personal  supervi- 
sion. At  the  bottom  of  the  vessel  is  a  thick  glass  plate  which  prevents  the 
thermometer  bulb  and  capillary  tubes,  which  rest  upon  it,  from  being  ex- 
posed to  heat  transmitted  directly  from  the  bottom  of  the  vessel.  To  further 
guard  against  this  I  am  in  the  habit  of  applying  the  flame  to  the  sides  of  the 
vessel,  and  a  uniform  temperature  throughout  the  bath  is  maintained  by 
frequent  stirring  with  a  glass  rod.  It  is  impossible  to  avoid  slight  variations, 
but  by  keeping  my  eye  upon  the  thermometer  throughout  the  experiment 
I  have  kept  these  within  very  narrow  limits.  ...  No  attempt  has  been  made 
to  fix  the  thermal  death-point  within  narrower  limits  than  2°  C.,  and  in  the 
table  the  lowest  temperature  is  given  which  has  been  found,  in  the  experi- 
ments made,  to  destroy  all  of  the  microorganisms  in  the  material  subjected 
to  the  test.  No  doubt  more  extended  experiments  would  result,  in  some  in- 
stances, in  a  reduction  of  the  temperature  given  as  the  thermal  death-point 
for  a  degree  or  more.  But  the  results  as  stated  are  sufficiently  accurate  for 
all  practical  purposes." ' 

The  results  obtained  in  these  experiments,  for  non-sporebearing 
bacteria,  are  given  in  the  following  table.  The  time  of  exposure 
was  ten  minutes,  except  for  the  cholera  spirillum  and  the  cheese  spi- 
rillum of  Deneke. 

THERMAL   DEATH-POINT   OF  BACTERIA.. 


Centigrade. 

Fahrenheit. 

52° 

125.6°  (4  m  ) 

52 

125.6    (4m.) 

Spirillum  Finklcr-  Prior                                .         

50 

122 

Bacillus  typlii  abdominalis                    

56 

138  8 

58 

136.4 

58 

136.4 

62 

143.6 

62 

143.6 

56 

132.8 

54 

129.2 

56 

132.8 

58 

136.4 

58 

136.4 

54 

129.2 

54 

129.2 

Bacillus  acidi  lactici  

56 

132.8 

Stapliylococcus  pyosrenes  aureus         ••  . 

58 

136.4 

Staphylococcus  pyo^enes  citreus  

62 

143.6 

Stapliylococcus  pyo(rcnes  albus 

62 

143.6 

Streptococcus  pyosrenes  ...             .... 

54 

1292 

58 

136.4 

Micrococcus  PastGuri         .  - 

52 

125.6 

Sarcina  lutea  

64 

147.2 

62 

143.6 

The  following  determinations  of  the  thermal  death-point  of  path- 

1  Quoted  from  the  Report  of  the  Committee  on  Disinfectants  of  the  American  Pub- 
lic Health  Association,  pages  136  and  152. 


152  INFLUENCE  OF  PHYSICAL  AGENTS. 

ogenic  organisms  have  been  made  by  the  authors  named  :  Bacillus 
anthracis  (Chauveau),  54°  C.  ;  Bacillus  mallei— the  bacillus  of  glan- 
ders—(Loffler),  55°  C.,  Bacillus  gallinarum — micrococcus  of  fowl 
cholera— (Salmon),  56°  C.  ;  Bacillus  of  diphtheria  (Loffler),  60°  C. 

In  the  writer's  experiments  the  micrococcus  of  gonorrhoea  was 
apparently  killed  by  exposure  for  ten  minutes  to  a  temperature  of 
60°  C. 

"Somegonorrhceal  pus  from  a  recent  case  which  had  not  undergone 
treatment  was  collected  for  me  by  my  friend  Dr.  Rohe  in  the  capillary 
glass  tubes  heretofore  described.  A  microscopical  examination  of  stained 
cover-glass  preparations  showed  that  this  pus  contained  numerous  4  gono- 
cocci '  in  the  interior  of  the  cells.  Two  of  the  capillary  tubes  were  placed 
in  a  water  bath  maintained  at  60°  C.  for  ten  minutes.  The  pus  was  then 
forced  out  upon  two  pledgets  of  cotton  wet  with  distilled  water.  Two 
healthy  men  nad  consented  to  submit  to  the  experiment,  and  one  of  these 
bits  of  cotton  was  introduced  into  the  urethra  of  each  and  left  in  situ  for 
half  an  hour.  As  anticipated,  the  result  was  entirely  negative.  For  obvi- 
ous reasons  no  control  experiment  was  made  to  fix  the  thermal  death-point 
within  narrower  limits. 

"  In  connection  with  these  experiments  upon  the  thermal  death-point  of 
known  pathogenic  organisms,  it  is  of  interest  to  inquire  whether  the  viru- 
lence of  infectious  material,  in  which  it  has  not  been  demonstrated  that  this 
virulence  is  due  to  a  microorganism,  is  destroyed  by  a  correspondingly  low 
temperature.  Evidently,  if  this  proves  to  be  the  case,  it  will  be  a  strong 
argument  in  favor  of  the  view  that  we  have  to  deal  with  a  microorganism 
in  these  diseases  also.  We  have  experimental  proof  that  a  large  number  of 
pathogenic  organisms  are  killed  by  exposure  for  ten  minutes  to  a  tempera- 
ture of  55°  to  60°  C.  But,  so  far  as  I  am  aware,  this  low  temperature  would 
not  be  likely  to  destroy  any  of  the  poisonous  chemical  products  which  might 
be  supposed  to  be  the  cause  of  infective  virulence,  leaving  aside  the  fact  that 
such  chemical  products  have  no  power  of  self-multiplication,  and,  there- 
fore, could  not  be  the  independent  cause  of  an  infectious  disease. ] 

44  Vaccine  Virus. — Carstens  and  Coert  have  experimented  upon  the  tem- 
perature required  to  destroy  the  potency  of  vaccine  virus.  In  a  paper  read 
at  the  International  Medical  Congress  in  1879  they  report,  as  a  result  of 
their  experiments,  that  the  maximum  degree  of  heat  to  which  fresh  vaccine 
virus  can  be  exposed  without  losing  its  virulence  probably  varies  between 
52°  and  54*  C.  Fresh  animal  vaccine  heated  to  52°  C.  for  thirty  minutes 
•does  not  lose  its  virulence.  Fresh  animal  vaccine  heated  to  54.5 5  for  thirty 
minutes  loses  its  virulence. 

4 'Rinderpest.—  According  to  Semmer  and  Raupach,  exposure  for  ten 
minutes  to  a  temperature  of  55°  C.  destroys  the  virulence  of  the  infectious 
material  in  this  disease. 

**  Sheep-pox.— The  authors  last  mentioned  have  also  found  that  the  same 
temperature— 55°  C.  for  ten  minutes — destroys  the  virulence  of  the  blood  of 
an  animal  dead  from  sheep-pox. 

'•  //Y-//-O,,/,, ,/,,,,  |>rsinii-  t«>  li\  tli<>  tlnM-mal  <l«>atli-j>oint  of  the  virus  of 
lydrophobia,  I  obtained,  through  the  kindness  of  Dr.  H.  C.  Ernst,  a  rabbit 
which  had  been  inoculated,  by  the  method  of  trephining,  with  material 

lch  came  originally  from  Pasteur's  laboratory.     The  rabbit  sent  me 
showed  the  first  symptom  of  paralytic  rabies  on  the  eighth  day  after  inocu- 
It  died  on  the  eleventh  day  (March  2d,  1887),  and  I  at  once  pro- 
MMM  to  i,,ak«>  th«-  f.,Ilmvii,ir  ••xp.'miiont  : 

A  portion  of  the  medulla  was  removed  and  thoroughly  mixed  with 

K,?lfl?  I?18,,?*8  written  Brieger  has  isolated  a  toxalbumin  from  cultures  of  the 
diphtheria  bacillus  which  is  destroyed  by  a  temperature  of  60°  C.,  but  resists  50°. 


INFLUENCE   OF   PHYSICAL   AGENTS.  153 

sterilized  water.  The  milky  emulsion  was  introduced  into  four  capillary 
tubes,  such  as  had  been  used  in  my  experiments  heretofore  recorded.  Two 
of  these  tubes  were  then  placed  for  ten  minutes  in  a  water  bath,  the  tem- 
perature of  which  was  maintained  at  60°  C.  Four  rabbits  were  now  inocu- 
lated by  trephining,  two  with  the  material  exposed  to  60°  C.  for  ten  min- 
utes, and  two  with  the  same  material  from  the  capillary  tube  not  so  exposed. 
The  result  was  as  definite  and  satisfactory  as  possible.  The  two  control 
rabbits  were  taken  sick,  one  on  March  10th  and  one  on  the  llth  ;  both  died 
with  the  characteristic  symptoms  of  paralytic  rabies  on  the  third  day.  The 
two  rabbits  inoculated  with  material  exposed  to  60°  C.  remained  in  perfect 
health.  On  the  26th  of  March  one  of  these  rabbits  was  again  inoculated, 
by  trephining,  with  material  from  the  medulla  of  a  rabbit  just  dead  from 
hydrophobia.  This  rabbit  died  from  paralytic  rabies  on  the  8th  of  April. 
Its  companion  remains  in  perfect  health. 

"A  second  experiment  was  made  in  the  same  way  on  the  14th  of  March. 
Two  rabbits  were  inoculated  with  material  exposed  for  ten  minutes  to  a 
temperature  of  50°  C. ;  two  with  material  exposed  to  55°  C. ;  and  two  con- 
trol rabbits  with  material  not  so  exposed.  One  of  the  rabbits  inoculated 
with  material  exposed  to  50°  C.,  and  one  of  the  control  rabbits,  died  on  the 
25th;  the  other  rabbit  inoculated  with  the  material  exposed  to  50°,  the  other 
control,  and  one  inoculated  with  material  exposed  to  55°,  on  the  26th.  The 
second  rabbit  inoculated  with  material  exposed  to  55°  died  five  days  later 
with  the  characteristic  symptoms  of  the  disease.  These  experiments  show, 
then,  that  the  virus  of  hydrophobia  is  destroyed  by  a  temperature  of  60°  C., 
and  that  55°  C.  fails  to  destroy  it,  the  time  of  exposure  being  ten  minutes."1 

The  experimental  data  given  show  that  the  pathogenic  bacteria 
tested  and  different  kinds  of  virus  are  all  killed  by  a  temperature  of 
60°  C.  or  below ;  some,  like  the  cholera  spirillum  and  Micrococcus 
pneumonias  crouposse,  failing  to  grow  after  exposure  to  as  low  a  tem- 
perature as  52°  C.  for  four  minutes.  By  extending  the  time  a  still 
lower  temperature  will  effect  the  same  result.  Thus,  according  to 
Chauveau,  the  anthrax  bacillus  is  killed  by  twenty  minutes7  exposure 
to  a  temperature  of  50°  C. ;  and  Brieger  sterilizes  cultures  of  the 
diphtheria  bacillus,  to  obtain  the  soluble  toxalbumin  produced  in 
them,  by  exposure  for  several  hours  to  50°  C.  A  temperature  of  60° 
has  been  found  to  decompose  the  toxalbumin.  The  non-pathogenic 
bacteria  tested  have,  as  a  rule,  a  higher  thermal  death-point — 58°  C. 
for  Bacillus  prodigiosus,  64°  C.  for  Sarcina  lutea,  etc. 

It  is  a  remarkable  fact  that  certain  bacteria  not  only  are  not  de- 
stroyed at  higher  temperatures  than  this,  but  are  able  to  multiply  at 
a  temperature  of  65°  to  70°  C.  Thus  Miquel,  in  1881,  found  in  the 
waters  of  the  Seine  a  motionless  bacillus  which  grew  luxuriantly  in 
bouillon  at  a  temperature  of  69°  to  70°  C.  Van  Tieghem  has  also 
cultivated  several  different  species  at  about  the  same  temperature, 
and  more  recently  Globig  has  obtained  from  the  soil  several  species 
which  grow  at  temperatures  ranging  from  50°  to  70°  C. 

The  resisting  power  of  spores  to  heat  also  varies  in  different  spe- 
cies ;  but  the  spores  of  known  pathogenic  bacteria  are  quickly  de- 
stroyed by  a  temperature  of  100°  C.  (212°  F.).     In  the  writer's  experi- 
1  Report  of  the  Committee  on  Disinfectants  (op.  cit.),  p.  147. 


l.VJ  INFLUENCE  OF  PHYSICAL  AGENTS. 

ments  the  spores  of  Bacillus  anthracis  and  of  Bacillus  alvei  failed  to 
grow  after  exposure  to  a  temperature  of  100°  C.  for  four  minutes, 
and  only  a  few  colonies  developed  after  two  minutes'  exposure  to  this 
temperature.  The  thermal  death-point  of  spores  of  the  "  wurtzel  ba- 
cillus "  and  of  Bacillus  butyricus  (of  Hueppe)  was  the  same — 100°  C. 
for  four  minutes. 

Schill  and  Fischer,  in  1884,  made  a  number  of  experiments  to  de- 
termine the  thermal  death-point  of  Bacillus  tuberculosis.  They 
found  that  five  minutes'  exposure  to  a  temperature  of  100°  C.  in 
steam  destroyed  the  vitality  of  the  bacillus  in  sputum  in  five  min- 
utes. When  the  time  was  reduced  to  two  minutes  a  negative  result 
from  inoculation  was  obtained  in  two  guinea-pigs,  but  one  inoculated 
at  the  same  time  became  tuberculous.  My  own  experiments  and 
those  of  Yersin,  made  since,  lead  me  to  think  that  there  may  have 
been  some  cause  of  error  in  this  experiment  of  Schill  and  Fischer, 
and  that  the  thermal  death-point  of  the  spores  of  Bacillus  tuber- 
culosis is  considerably  below  the  boiling  point  of  water.  I  inoculated 
guinea-pigs  with  tuberculous  sputum  subjected  for  ten  minutes  to 
the  following  temperatures :  50°,  60°,  70°,  80°,  90°  C.  The  animal 
inoculated  with  material  exposed  to  50°  died  from  tuberculosis  at  the 
end  of  seven  weeks.  None  of  the  others  developed  tuberculosis. 

Yersin  exposed  an  old  culture  in  glycerin  bouillon,  in  which  many 
of  the  bacilli  contained  spores—"  tres  nettes" — to  the  following  tem- 
peratures :  55°,  60°,  65°,  70°,  75°,  80°,  85°,  90°,  100°  C.  "  At  the  end  of 
ten  days  the  bacilli  heated  to  55°  gave  a  culture  in  glycerin  bouillon ; 
those  exposed  to  60°  grew  after  twenty-two  days ;  none  of  the 
bacilli  heated  above  70°  gave  any  development.  This  experiment, 
repeated  a  great  number  of  times,  has  always  given  us  the  same  re- 
sult." Voelsh,  who  has  studied  the  same  question,  reports  as  the 
result  of  his  experiments  that  the  tubercle  bacillus  in  sputum  was 
not  destroyed  by  heating  to  100°  C.  Further  experiments  will  be  re- 
quired to  reconcile  these  contradictory  results. 

While  the  spores  of  the  pathogenic  bacteria  mentioned  are  de- 
stroyed by  the  boiling  point  of  water  within  a  few  minutes,  certain 
non-pathogenic  species  resist  this  temperature  for  hours.  Thus 
Qlobig  obtained  a  bacillus  from  the  soil  the  spores  of  which  required 
five  and  one-half  to  six  hours'  exposure  to  streaming  steam  for  their 
destruction.  These  spores  survived  exposure  for  three-quarters  of  an 
hour  in  st. -am  under  pressure  at  from  109°  to  113°  C.  They  were  de- 
stroyed, however,  by  exposure  for  twenty-five  minutes  in  steam  at 
113°  to  11U°,  and  in  two  minutes  at  127°. 

In  the  practical  application  of  steam  for  disinfecting  purposes  it 
must  be  remembered  that,  while  steam  under  pressure  is  more  effec- 
tive than  streaming  steam,  it  is  scarcely  necessary  to  give  it  the  pre- 


INFLUENCE   OF   PHYSICAL  AGENTS.  155 

ference,  in  view  of  the  fact  that  all  known  pathogenic  bacteria  and 
their  spores  are  quickly  destroyed  by  the  temperature  of  boiling 
water  ;  and  also  that  superheated  steam  is  less  effective  than  moist 
steam.  When  confined  steam  in  pJpes  is  ' '  superheated  "  it  has  about 
the  same  germicidal  power  as  hot  dry  air  at  the  same  temperature. 
This  is  shown  by  the  experiments  of  Esmarch,  who  found  that  an- 
thrax spores  were  killed  in  streaming  steam  in  four  minutes,  but 
were  not  killed  in  the  same  time  by  superheated  steam  at  a,  tempera- 
ture of  141°  C. 

Desiccation. — Cultures  of  bacteria  kept  in  a  moist  condition  re- 
tain their  vitality  for  a  considerable  time,  which  varies  greatly  with 
different  species.  The  writer  has  found  that  a  culture  of  the  typhoid 
bacillus  preserved  in  a  hermetically  sealed  glass  tube  retained  its 
vitality  for  eighteen  months,  as  did  also  Bacillus  prodigiosus,  Bacil- 
lus cavicida,  and  some  others.  According  to  Kitasato,  the  cholera 
spirillum  may  be  preserved  in  a  moist  state  for  seven  months  ;  other 
bacteria  die  out  in  a  month  or  two,  but,  as  a  rule,  vitality  is  preserved 
for  several  months  at  least. 

Spores  in  a  desiccated  condition  preserve  their  vitality  for  a 
great  length  of  time.  But  desiccation  is  quickly  fatal  to  some  of  the 
pathogenic  bacteria,  and  especially  so  to  the  cholera  spirillum.  Koch, 
in  his  earlier  experiments,  found  that  his  " comma  bacillus"  did  not 
grow  after  being  dried  upon  a  cover  glass  for  three  hours.  Kitasato, 
in  experiments  made  since,  found  that  a  bouillon  culture  dried  upon 
a  thin  glass  cover  was  incapable  of  development  after  three  hours' 
time,  but  that  cultures  in  nutrient  agar  or  gelatin  survived  for  two 
days,  probably  on  account  of  the  thicker  layer  formed  and  the  longer 
time  required  for  complete  desiccation.  Pfuhl  has  found  that  the 
typhoid  bacillus  dried  upon  a  cover  glass  retains  its  vitality  for 
eight  to  ten  weeks,  and  Lofner  states  that  the  diphtheria  bacillus  re- 
sists desiccation  for  four  or  five  months.  Cadeac  and  Malet  pro- 
duced tuberculosis  in  guinea-pigs  by  injecting  material  from  the 
lung  of  a  tuberculous  cow  which  had  been  kept  in  the  form  of  a  dried 
powder  for  nearly  five  months  ;  at  a  later  date  the  virulence  was 
lost. 

Light. — Downes  and  Blunt,  in  a  communication  made  to  the 
Royal  Society  of  London  in  1877,  first  called  attention  to  the  fact  that 
light  has  an  injurious  effect  upon  bacteria,  and  that  cultures  may  be 
sterilized  by  exposure  to  direct  sunlight. 

Tyndall,  in  experiments  made  in  the  clear  sunlight  of  the  Alps, 
verified  the  fact  that  the  development  of  bacteria  was  restrained  in 
cultures  during  their  exposure,  but  failed  to  obtain  evidence  that 
vitality  was  destroyed. 

In  1885  Duclaux  took  up  the  subject  with  pure  cultures  of  various 


156  INFLUENCE  OF  PHYSICAL  AGENTS. 

bacteria,  and  showed  that  by  prolonged  exposure  to  direct  sunlight  the 
spores  of  various  bacilli  lose  their  capacity  to  germinate.  About  the 
same  time  Arloing  published  his  researches  upon  the  influence  of 
light  upon  the  development  of  anthrax  spores.  He  found  that  the 
anthrax  bacillus  was  not  restrained  in  its  growth  by  diffused  lamp- 
light, but  its  growth  was  retarded  by  an  intense  gaslight.  Spore 
formation  was  more  abundant  in  darkness  than  in  red  light,  and  more 
abundant  in  red  than  in  white  light.  When  a  screen  was  interposed 
between  the  culture  and  the  source  of  light,  consisting  of  an  aqueous 
solution  of  hgematoglobin,  the  growth  of  the  bacilli  and  of  spores  was 
much  more  luxuriant  than  in  white  light.  In  yellow  light  it  was  less 
abundant  than  in  red.  The  blue  and  violet  rays  were  still  less  favor- 
able for  the  growth  of  the  bacillus  and  the  development  of  spores. 
The  pathogenic  power  of  cultures  was  not  especially  influenced  by 
exposure  to  white  gaslight.  In  subsequent  experiments  with  sun- 
light Arloing  found  that  two  hours  of  exposure  to  the  July  sun  suf- 
ficed to  destroy  the  vitality  of  anthrax  spores,  but  that  a  considerably 
longer  exposure  (twenty-six  to  thirty  hours)  was  necessary  when  the 
spores  had  been  allowed  to  germinate  in  a  suitable  culture  medium. 
Cultures  which  were  not  exposed  long  enough  to  destroy  the  vitality 
of  the  bacilli  were  retarded  in  their  growth,  and  subsequent  exposure 
for  a  shorter  time  (nine  to  ten  hours)  completely  sterilized  them. 
Cultures  which  were  weakened  in  their  reproductive  energy  by  ex- 
posure to  sunlight  were  also  "attenuated"  as  to  their  pathogenic 
power  and  could  be  used  as  a  vaccine  in  protective  inoculations.  Ac- 
cording to  Arloing,  the  effect  produced  results  from  the  action  of  the 
full  sunlight  and  cannot  be  obtained  by  the  use  of  monochromatic 
light. 

The  experiments  of  Strauss  seemed  to  give  support  to  the  view 
advanced  by  Nocard  that  in  Arloing's  experiments  spores  did  not 
really  exhibit  a  less  degree  of  resisting  power  than  the  vegetating 
Iwicilli,  but  that  in  fact  they  commenced  to  vegetate  before  they  were 
killed.  Strauss  placed  anthrax  spores  in  sterilized  distilled  water  and 
in  txmillnn,  and  found  that,  under  the  same  conditions  of  exposure, 
the  bouillon  cultures  were  sterilized  in  direct  sunlight  in  nine 
hours,  while  the  spores  suspended  in  distilled  water  grew  when  trans- 
ferred to  a  suitable  medium.  This  was  accounted  for  on  the  suppo- 
>iti«>n  that  the  bouillon  furnishes  the  necessary  pabulum  for  the  de- 
velopment of  the  spores  and  that  distilled  water  does  not. 

Arloing  combats  this  view  and  has  published  additional  experi- 
ments which  seem  to  disprove  it.  He  placed  small  flasks  containing 
anthrax  spores  in  bouillon  in  the  direct  rays  of  the  sun  in  February. 
Some  of  the  flasks  were  placed  upon  a  block  of  ice  which  reduced  the 
temperature  to  4°  C. ;  the  others  were  not  so  placed,  and  the  tempe- 


INFLUENCE   OF   PHYSICAL   AGENTS.  157 

rature,  in  the  open  air  where  all  were  exposed,  was  11°  C.  All  of  the 
spores  failed  to  grow  after  an  exposure  of  four  hours.  When  exposed 
in  water  the  time  of  exposure  was  longer. 

Roux  has  shown  that  the  light  also  has  an  effect  upon  the  culture 
medium,  and  that  sterilized  bouillon  which  has  been  exposed  to  direct 
sunlight  for  some  hours  restrains  the  development  of  anthrax  spores 
subsequently  introduced  into  it,  but  not  of  the  growing  bacilli.  His 
experiments  show  that  access  of  oxygen  is  a  necessary  factor  in  the 
sterilization  of  cultures  by  sunlight. 

In  the  experiments  of  Momont  (1892)  dry  anthrax  spores  were 
found  to  resist  the  action  of  light  for  a  long  time,  but  moist  spores, 
freely  exposed  to  the  air,  failed  to  grow  after  forty-four  hours'  ex- 
posure to  sunlight.  In  the  absence  of  spores,  anthrax  bacilli  in  a 
moist  condition,  when  freely  exposed  to  the  air,  failed  to  grow  after 
exposure  to  sunlight  for  half  an  hour  to  two  hours  ;  but  in  the  ab- 
sence of  air  the  same  bacilli  were  not  destroyed  at  the  end  of  fifty 
hours'  exposure. 

Geisler  (1892),  in  experiments  made  upon  the  typhoid  bacillus, 
found  that  all  portions  of  the  solar  spectrum  except  the  red  rays  ex- 
ercised a  restraining  influence  upon  the  development  of  this  bacillus. 
The  electric  light  gave  a  similar  result.  The  most  decided  effect  was 
produced  by  rays  from  the  violet  end  of  the  spectrum.  The  restrain- 
ing influence  appears,  fro.ni  the  researches  of  Geisler,  not  to  be  due 
solely  to  the  direct  action  of  light  upon  the  development  of  the 
bacilli,  but  also  to  changes  induced  in  the  gelatin  culture  medium 
employed  in  his  experiments. 

In  his  address  before  the  International  Medical  Congress  of  Berlin, 
1890,  Koch  states  that  the  tubercle  bacillus  is  killed  by  the  action  of 
direct  sunlight  in  a  time  varying  from  a  few  minutes  to  several  hours, 
depending  upon  the  thickness  of  the  layer  exposed.  Diffused  day- 
light also  has  the  same  effect,  although  a  considerably  longer  time  of 
exposure  is  required — when  placed  close  to  a  window,  from  five  to 
seven  days. 

Dieudonne  (1894),  in  experiments  upon  Bacillus  prodigiosus  and 
Bacillus  fluorescens  putidus,  found  that  direct  sunlight  in  March, 
July,  and  August  killed  these  bacilli  in  one  and  one-half  hours,  in 
November  in  two  and  one-half  hours.  Diffuse  daylight  in  March 
and  July  restrained  development  after  three  and  one-half  hours'  ex- 
posure (in  November  four  and  one-half  hours),  and  completely  de- 
stroyed vitality  in  from  five  to  six  hours. 

Ward's  experiments  (1892-1894)  show  that  the  blue  and  violet 
rays  have  decided  germicidal  power,  while  the  rays  at  the  red  end  of 
the  spectrum  are  comparatively  inert.  This  corresponds  with  results 
previously  reported  by  Arloing. 


158  INFLUENCE  OF  PHYSICAL  AGENTS. 

In  the  writer's  experiments  on  the  cholera  spirillum  (1892)  test 
tubes,  containing  sterile  bouillon  inoculated  with  one  or  two  ose  of  a 
pure  culture,  were  sterilized  by  two  hours'  exposure  to  direct  sunlight 
(in  December). 

Dieudonne  (1894)  found  that  the  electric  arc  light  destroyed  his 
test  organisms  (Bacillus  prodigiosusand  Bacillus  fluorescens  putidus) 
in  eight  hours.  The  same  result  was  accomplished  by  the  incandes- 
cent light  in  eleven  hours. 

In  view  of  these  facts  we  may  conclude,  with  Duclaux,  that  sun- 
light is  one  of  the  most  potent  and  one  of  the  cheapest  agents  for  the 
destruction  of  pathogenic  bacteria,  and  that  its  use  for  this  purpose  is 
to  be  remembered  in  making  practical  hygienic  recommendations. 
The  popular  idea  that  the  exposure  of  infected  articles  of  clothing 
and  bedding  in  the  sun  is  a  useful  sanitary  precaution  is  fully  sus- 
tained by  the  experimental  data  relating  to  the  action  of  heat,  desic- 
cation, and  sunlight. 

Electricity. — Cohn  and  Mendelssohn,  in  1879,  attempted  to  de- 
termine the  effect  of  the  galvanic  current  upon  bacteria.  Cultures 
were  placed  in  U  -tubes  through  which  a  constant  current  was  passed. 
A  feeble  current  was  found  to  be  without  effect.  A  strong  current 
from  two  elements,  maintained  for  twenty-four  hours,  restrained  de- 
velopment in  the  vicinity  of  the  positive  pole,  but  this  was  probably 
due  to  the  highly  acid  reaction  which  the  culture  liquid  acquired. 
When  a  current  from  five  elements  was  used  for  twenty-four  hours 
the  liquid  was  sterilized,  but  this  may  have  been  due  to  the  decided 
changes  produced  in  the  chemical  composition  of  the  culture  liquid 
rather  than  to  the  direct  action  of  the  galvanic  current. 

The  same  may  be  said  of  the  similar  results  obtained  in  later  ex- 
periments by  Apostoli  and  Laquerriere,  and  by  Prochownick  and 
Spaeth.  The  last-mentioned  investigators  found  that  the  positive  pole 
had  a  more  decided  effect  than  the  negative,  and  that  the  effect  de- 
pended upon  the  intensity  and  duration  of  the  current.  A  current  of 
lit  ty  milliamperes  passed  for  a  quarter  of  an  hour  did  not  kill  Staphy- 
lococcus  pyogenes  aureus,  but  a  current  of  sixty  milliamperes  main- 
tained for  the  same  time  did.  The  spores  of  Bacillus  anthracis 
required  a  current  of  two  hundred  to  two  hundred  and  thirty  milli- 
amperes during  an  hour  or  two.  In  these  experiments  the  cultures 
in  gelatin  were  attached  to  the  strips  of  platinum  serving  as  the  two 
poles,  and  these  were  immersed  in  a  solution  of  sodium  chloride.  As 
chlorine  was  disengaged  at  the  positive  pole,  the  germicidal  action  is 
attributed  to  this  gas  rather  than  to  the  direct  action  of  the  current 
upon  the  living  microorganisms. 

The  more  recent  researches  of  Spilker  and  Gottstein,  made  with 
an  induction  current  from  a  dynamo  machine,  are  more  valuable  in 


INFLUENCE   OF   PHYSICAL   AGENTS.  159 

estimating  the  power  of  this  agent  to  destroy  the  vitality  of  bacteria. 
The  current  was  passed  through  a  spiral  wire  which  was  wrapped 
around  a  test  tube  of  glass,  containing  the  microorganism  to  be  tested, 
suspended  in  distilled  water.  In  a  first  experiment  Bacillus  prodigi- 
osus,  suspended  in  sterilized  distilled  water  and  contained  in  test 
tubes  having  a  capacity  of  two  hundred  and  fifty  cubic  centimetres, 
was  subjected  to  a  current  having  an  energy  of  2.5  amperes  X  1.25 
volts  for  twenty-four  hours.  The  temperature  did  not  go  above 
30°  C.  No  development  occurred  when  the  microorganism  tested 
was  subsequently  planted  in  nutrient  gelatin.  Further  experiments 
gave  a  similar  result.  It  was  found  that  stronger  currents  were 
effective  in  shorter  time ;  but  in  no  case  was  sterilization  effected  in 
less  than  an  hour. 

Pressure. — D'Arsonval  and  Charrin  (1894)  submitted  a  culture 
of  Bacillus  pyocyaneus  to  a  pressure  of  fifty  atmospheres,  under  car- 
bon dioxide.  At  the  end  of  four  hours  cultures  could  still  be  ob- 
tained, but  the  bacillus  had  lost  its  power  of  pigment  production.  A 
few  colonies  were  developed  after  six  hours'  exposure  to  this  pressure ; 
but  after  twenty-four  hours  no  development  occurred. 

Agitation. — Meltzer  (1894)  has  shown  that  the  vitality  of  bacteria 
is  destroyed  by  protracted  and  violent  shaking,  which  causes  a  molec- 
ular disintegration  of  the  cells. 


VII. 
ANTISEPTICS  AND  DISINFECTANTS. 

GENERAL  ACCOUNT  OF  THE  ACTION  OF. 

THE  term  antiseptic  is  used  by  some  authors  to  designate  an 
which  destroys  the  vitality  of  the  microorganisms  which  pro- 
duce septic  decomposition,  and  others  of  the  same  class.  We  prefer 
to  restrict  the  use  of  the  term  to  those  agents  which  restrain  the  de- 
velopment of  such  microorganisms  without  destroying  their  vitality. 
The  complete  destruction  of  vitality  is  effected  by  germicides  or  dis- 
infectants. Material  containing  the  germs  of  infectious  diseases  is 
infectious  material,  and  we  disinfect  it  by  the  use  of  agents  which 
destroy  the  living  disease  germs  or  pathogenic  bacteria  which  give 
it  its  infecting  power.  Such  an  agent  is  a  disinfectant.  But  we  ex- 
tend the  use  of  this  term  to  germicides  in  general — that  is,  to  those 
agents  which  kill  non-pathogenic  bacteria  as  well  as  to  those  which 
destroy  disease  germs.  All  disinfectants  are  also  antiseptics,  for 
agents  which  destroy  the  vitality  of  the  bacteria  of  putrefaction  ar- 
rest the  putrefactive  process  ;  and  these  agents,  in  less  amount  than 
is  required  to  completely  destroy  vitality,  arrest  growth  and  thus 
act  as  antiseptics.  But  all  antiseptics  are  not  germicides.  Thus  a 
concentrated  solution  of  salt  or  of  sugar  will  prevent  the  putrefac- 
tive decomposition  of  organic  material,  animal  or  vegetable  ;  but  these 
agents  do  in»t  destroy  the  vitality  of  the  germs  of  putrefaction.  In 
a  certain  degree  of  concentration  they  are  antiseptics  and  are  largely 
u>.-.l  f.,r  the  preservation  of  meats  and  vegetables.  In  the  same  way 
many  mineral  salts  in  solutions  of  various  strengths  act  as  antisep- 
tics, and  some  of  these  in  still  stronger  solutions  are  disinfectants. 
Thus  mercuric  chloride,  when  introduced  into  a  culture  solution  in 
the  proportion  of  1  :  300,000,  will  restrain  the  development  of  anthrax 
spores,  but  to  insure  the  destruction  of  these  spores  a  solution  of 
1  : 1,000  must  be  used.  As  a  rule,  the  difference  between  restraining 
action — antiseptic— and  germicidal  power — disinfectant — is  not  so 
great  as  this.  We  give  below  some  recent  determinations  by  Boer 
which  illustrate  this  point,  the  test  organism  being  the  bacillus  of 
typhoid  fever  in  a  culture  in  bouillon  twenty-four  hours  old  : 


ANTISEPTICS   AND   DISINFECTANTS. 


Restrains. 

Kills. 

1    2100 

1  -300 

1  :  1550 

1  •  500 

Silver  nitrate  

1  :  50000 

1  -4000 

1  :  6000 

1  •  250 

Carbolic  acid  ....   

1  :400 

1  -200 

Method  of  Determining  Antiseptic  Value. — To  determine  the 
restraining  or  antiseptic  power  of  an  agent  for  a  particular  micro- 
organism, the  agent  is  dissolved  in  a  definite  proportion  in  a  suitable 
culture  medium,  which  is  then  inoculated  with  a  pure  culture  of  the 
test  organism  and  placed  in  favorable  circumstances — as  to  tempera- 
ture— for  its  growth.  At  the  same  time  a  control  experiment  is 
made  by  placing  another  portion  of  the  same  culture  medium,  inocu- 
lated with  the  same  microorganism,  in  the  same  conditions,  but  with- 
out the  addition  of  the  antiseptic  agent.  If  development  occurs  in 
the  control  experiment  and  not  in  the  culture  medium  containing 
the  antiseptic,  the  failure  to  grow  must  be  attributed  to  the  presence 
of  this  agent.  Having  made  a  preliminary  experiment,  we  are 
guided  by  the  result  in  further  experiments  to  determine  the  exact 
amount  required  to  restrain  development  under  the  same  conditions. 
Or  we  may  make  a  series  of  experiments  in  the  first  instance.  The 
problem  being,  for  example,  to  determine  the  antiseptic  value  of 
carbolic  acid  for  the  typhoid  bacillus,  we  may  add  this  agent  to  a 
definite  amount  of  bouillon  in  test  tubes  in  the  proportion  of  1  : 100, 
1  : 200,  1  : 300,  1  : 400,  1  : 500.  In  experiments  with  volatile  agents 
the  bouillon,  in  test  tubes  or  small  flasks,  must  be  sterilized  in  ad- 
vance, and  the  antiseptic  agent  introduced  by  means  of  a  sterilized 
pipette  with  great  care  to  prevent  the  accidental  contamination  of 
the  nutrient  medium.  In  experiments  with  non-volatile  agents  it  will 
be  best  to  sterilize  the  culture  medium  after  the  antiseptic  has  been 
added.  Next  we  inoculate  the  liquid  in  each  flask  with  a  pure  cul- 
ture of  the  test  organism.  The  flasks  are  then  placed  in  an  incubat- 
ing oven  at  35°  to  37°  C.  At  the  same  time  a  control,  not  containing 
any  carbolic  acid,  is  placed  in  the  oven.  At  the  end  of  twenty-four 
hours  the  control  will  be  found  to  be  clouded,  showing  an  abundant 
multiplication  of  the  bacillus.  Taking  the  result  of  Boer  above  given, 
we  would  expect  to  find  all  of  the  solutions  clear  except  that  contain- 
ing 1  :  500.  This  too  might  remain  clear  for  some  days  and  finally 
**  break  down/'  for  experience  shows  that  when  we  pass  the  point  at 
which  a  permanent  restraining  influence  is  exerted  there  may  be  a 
temporary  restraint  or  retardation  of  development.  For  this  reason 
we  must  continue  the  experiment  for  a  considerable  time — not  less 
11 


]62  ANTISEPTICS   AND   DISINFECTANTS. 

than  two  weeks.  Having  found  that  1  :400  and  below  prevents 
development,  and  1  . 500  does  not,  we  may  make  further  experiments 
to  determine  the  antiseptic  power  within  narrower  limits ;  but  this 
is  hardly  necessary  from  a  practical  point  of  view. 

In  these  experiments  the  result  will  be  influenced  by  several  cir- 
cumstances, as  follows  : 

(a)  By  the  composition  of  the  nutrient  medium.     This  is  a 
very  important  factor,  especially  in  determining  the  antiseptic  value 
of  certain  metallic  salts.     The  presence  of  a  considerable  quantity 
of  albumin,  for  example,  reduces  greatly  the   antiseptic  power  of 
mercuric  chloride,  silver  nitrate,  creolin,  etc.    The  presence  of  a  sub- 
stance chemically  incompatible,  as,  for  example,  sodium  chloride  in 
testing  nitrate  of  silver,  will  of  course  neutralize  antiseptic  action. 

(b)  The  nature  of  the  test  organism.     Within  certain  limits  an 
antiseptic  for  one  microorganism  of  this  class  restrains  the  devel- 
opment of  all,  but  there  are  wide  differences  in  the  ability  of  differ- 
ent species  to  grow  in  the  presence  of  different  chemical  agents. 
Some  grow  readily  in  the  presence  of  a  considerable  amount  of  free 
acid,  others  are  restrained  by  a  slightly  acid  reaction  of  the  medium 
in  which  they  are  placed.     The  Bacillus  acidi  lactici,  for  example, 
can  thrive  in  the  presence  of  a  considerable  amount  of  the  acid 
which  is  a  product  of  its  growth,  but  there  is  a  limit  to  its  power  of 
developing  in  the  presence  of  this  and  other  acids.     So,  too,   Mi- 
crococcus  urese,  which  causes  the  alkaline  fermentation  of  urine, 
grows  in  the  presence  of  a  considerable  amount  of  carbonate  of  am- 
monia, but  is  finally  restrained  in  its  growth  by  this  alkaline  salt. 
The  following  determinations  by  Boer  show  the  difference  in  the 
antiseptic  power  of  hydrochloric  acid  for  certain  pathogenic  bacte- 
ria :  Bacillus  of  anthrax  (without  spores),  1  : 3,400  ;  diphtheria  bacil- 
lus, 1  : 3,400  ;  glanders  bacillus,   1  :  700  ;  typhoid  bacillus,   1  :  2,100  ; 
cholera  spirillum,  1  :5,500.     It  will  be  noted  that  the  cholera  spiril- 
lum is  restrained  in  its  growth  by  about  one-eighth  the  amount  of 
hydrochloric  acid  which  is  required  to  prevent  the  development  of 
the  bacillus  of  glanders.     The  typhoid  bacillus  has  a  special  tole- 
rance for  carbolic  acid,  etc. 

(c)  The  temperature  at  which  the  experiment  is  made.     At 
the  temperature  most  favorable  for  growth  a  greater  proportion  of 
the  antiseptic  agent  is  required  than  at  unfavorable  temperatures- 
lower  or  higher. 

(d)  The  restraining  influence  for  spores  is  much  greater  than 
for  the  vegetative  form  of  bacteria. 

Methods  of  Determining  Germicide  Value.— The  disinfecting 
power  of  a  chemical  agent  is  determined  by  allowing  it  to  act  for  a 
given  time,  in  a  definite  proportion,  on  a  pure  culture  of  a  given 


ANTISEPTICS   AND   DISINFECTANTS.  163 

microorganism,  and  then  testing  the  question  of  loss  of  vitality  by 
culture  experiments  or  by  inoculations  of  infectious  disease  germs 
into  susceptible  animals. 

The  test  by  cultivation  is  the  most  reliable,  but  in  making  it 
several  points  must  be  kept  in  view.  Naturally  the  conditions  must 
be  such  as  are  favorable  for  the  growth  of  the  particular  microor- 
ganism which  serves  as  the  test ;  and  we  must  allow  a  considerable 
time  for  the  development  of  the  test  organism,  for  it  often  happens 
that  its  vital  activity  has  been  weakened  without  being  completely 
destroyed,  and  that  growth  will  occur  after  an  interval  of  several 
days,  while  in  the  control  experiment  it  has  perhaps  been  seen  at 
the  end  of  twenty-four  hours.  Another  most  important  point  is  the 
fact  that  some  of  the  disinfecting  agent  is  necessarily  carried  over 
with  the  test  organisms  when  these  are  transferred  to  a  nutrient 
medium  to  ascertain  whether  they  will  grow,  and  this  may  be  in 
sufficient  amount  to  restrain  their  development  and  lead  to  the  mis- 
taken inference  that  they  have  been  killed.  This  is  especially  true 
of  mercuric  chloride,  which  restrains  the  development  of  spores  in 
very  minute  amounts.  Spores  which  have  been  subjected  to  its  ac- 
tion in  comparatively  strong  solutions,  when  transferred  to  a  culture 
medium  may  fail  to  grow  because  of  the  restraining  influence  of 
the  mercuric  chloride  carried  over  at  the  same  time.  For  this  rea- 
son liquid  cultures  are  to  be  preferred  in  experiments  of  this  kind. 
When  the  test  organisms  are  planted  in  a  solid  culture  medium  the 
chemical  agent  is  left  associated  with  them  ;  in  a  liquid  culture,  on 
the  other  hand,  it  is  diluted,  and  the  microorganisms,  being  distri- 
buted through  the  nutrient  medium,  have  the  disinfecting  agent 
washed  from  their  surface.  In  the  case  of  mercuric  chloride,  how- 
ever, the  experiments  of  Geppert  show  that  the  agent  is  so  attached 
to  spores  which  have  been  subjected  to  its  action  that  ordinary 
washing  does  not  suffice.  Moreover,  spores  which  have  been  ex- 
posed to  the  action  of  mercuric  chloride  without  being  killed  are  re- 
strained in  their  growth  by  a  much  smaller  proportion  of  the  corro- 
sive sublimate  than  is  required  for  spores  not  so  exposed — according 
to  Geppert,  by  1  part  in  2,000,000.  Geppert  therefore  proposes,  in 
experiments  with  this  agent,  to  neutralize  the  mercuric  chloride 
which  remains  attached  to  the  test  organisms  by  washing  these  in 
a  solution  of  ammonium  sulphide,  by  which  the  sublimate  is  preci- 
pitated as  an  inert  sulphide. 

With  most  agents  simple  dilution  will  serve  the  purpose  of  pre- 
venting an  erroneous  inference  from  the  restraining  influence  of  the 
chemical  agent  being  tested.  If  we  carry,  by  means  of  a  platinum 
loop,  one  or  two  ose  into  five  to  ten  cubic  centimetres  of  bouillon, 
the  dilution  will  usually  be  beyond  the  restraining  influence  of  the 


1G4  ANTISEPTICS  AND   DISINFECTANTS. 

germicidal  agent ;  but  we  may  carry  the  dilution  still  further,  to  be 
on  the  side  of  safety,  by  inoculating  a  second  tube  containing  the 
same  amount  of  sterile  bouillon  from  the  first,  carrying  over  in  the 
same  way  one  or  two  ose.  We  will  still  be  very  sure  to  have  a 
considerable  number  of  the  microorganisms  to  test  the  question  of 
the  destruction  of  vitality.  Instead  of  bouillon  we  may  use  liquefied 
flesh-peptone-gelatin,  which  gives  us  the  same  advantage  as  to  dilu- 
tion of  the  disinfecting  agent ;  and  after  inoculating  two  tubes  as 
above  indicated,  we  may  make  Esmarch  roll  tubes  by  turning  them 
upon  a  block  of  ice.  The  development  of  colonies  will  show  that 
there  was  a  failure  to  disinfect ;  their  absence,  after  a  proper  inter- 
val, will  be  evidence  of  the  germicidal  action  of  the  agent  employed. 

Koch's  Method.— In  1881  Koch  published  his  extended  experi- 
ments made  to  determine  the  germicidal  power  of  various  chemical 
agents  as  tested  upon  anthrax  spores.  His  method  consisted  in  ex- 
posing silk  threads,  to  which  the  dried  spores  were  attached,  in  a 
solution  of  the  disinfecting  agent,  and  at  intervals  transferring  one 
of  these  threads  to  a  solid  culture  medium.  The  precaution  was 
taken  to  wash  the  thread  in  distilled  water  when  the  agent  tested  was 
supposed  to  be  likely  to  restrain  development.  In  these  experiments 
a  standard  solution  of  the  disinfecting  agent  was  used,  and  the  time 
of  exposure  was  varied  from  a  few  hours  to  many  days. 

The  Writer's  Method. — In  the  writer's  experiments,  made  in 
1 880  and  subsequently,  a  different  method  has  been  adopted.  The 
time  has  been  constant — usually  two  hours — and  the  object  has  been 
to  find  the  minimum  amount  of  various  chemical  agents  which 
would  destroy  the  test  organisms  in  this  time ;  and  instead  of  sub- 
jecting a  few  of  the  test  organisms  attached  to  a  silk  thread  to  the 
action  of  the  disinfecting  agent,  a  certain  quantity  of  a  recent  cul- 
ture— usually  five  cubic  centimetres — has  been  mixed  with  an  equal 
quantity*  of  a  standard  solution  of  the  germicidal  agent.  Thus  five 
cubic  centimetres  of  a  1  : 200  solution  of  carbolic  acid  would  be 
added  to  five  cubic  centimetres  of  a  recent  culture  of  the  typhoid 
bacillus,  for  example,  and  after  two  hours' contact  one  or  two  ose 
would  be  introduced  into  a  suitable  nutrient  medium  to  test  the 
question  <>t  disinfection.  In  the  case  given  the  result  obtained 
would  be  set  down  as  the  action  of  a  solution  of  carbolic  acid  in  the 
proportion  of  1 : 400,  for  the  1  :  200  solution  was  diluted  by  the  addi- 
i  u  equal  quantity  of  the  culture. 

other  experimenters  have  adopted  still  a  different  method.  In- 
stead of  using  a  considerable  and  definite  quantity  of  a  culture  con- 
taining the  test  organism,  they  introduce  one  or  two  ose  from  such 
a  culture  into  a  solution  containing  a  given  proportion  of  the  disin- 
fectant ;  then  after  exposure  for  a  given  time  the  nutrient  medium  is 
inoculated. 


ANTISEPTICS   AND    DISINFECTANTS. 


165 


These  different  methods  give  results  which  cannot  be  directly 
compared  one  with  another,  for  to  obtain  corresponding  results  we 
must  have  identical  conditions. 

Test  by  Inoculation  into  Susceptible  Animals. — In  testing  the 
action  of  disinfectants  upon  anthrax  spores  and  other  infectious  dis- 
ease germs,  we  may  inoculate  the  microorganisms,  after  exposure  to 
the  disinfectant,  into  a  susceptible  animal.  This  method  was  adopted 
by  the  writer  in  a  series  of  experiments  in  1881,  but  he  has  not  since 
employed  it,  for  reasons  set  forth  in  his  paper  giving  an  account  of 
these  experiments. 

"First.  The  test  organism  maybe  modified  as  regards  repro- 
ductive activity  without  being  killed;  and  in  this  case  a  modified  form 
of  disease  may  result  from  the  inoculation,  of  so  mild  a  character  as 
to  escape  observation.  Second.  An  animal  which  has  suffered  this 
modified  form  of  the  disease  enjoys  protection,  more  or  less  perfect, 
from  future  attacks,  and  if  used  for  a  subsequent  experiment  may, 
by  its  immunity  from  the  effects  of  the  pathogenic  test  organism, 
give  rise  to  the  mistaken  assumption  that  this  had  been  destroyed 
by  the  action  of  the  germicidal  agent  to  which  it  had  been  sub- 
jected."1 

In  experiments  to  determine  the  value  of  an  agent  as  a  disinfec- 
tant, no  matter  by  what  method,  the  following  conditions,  which  in- 
fluence the  result,  should  be  kept  in  view  : 

(a)  The  difference  in  vital  resisting  power  of  different  species 
of  bacteria.  As  a  rule,  the  pathogenic  species  have  rather  less  re- 
sisting power  than  the  common  saprophytes,  and  the  micrococci 
have  greater  resisting  power  than  many  of  the  bacilli.  The  differ- 
ence in  the  vital  resisting  power  of  some  of  the  best  known  patho- 
genic species  is  shown  in  the  following  table,  which  we  have  made 
up  from  determinations  made  by  Boer — cultures  in  bouillon  twenty- 
four  hours  old  ;  time  of  exposure,  two  hours. 


Hydrochloric 
Acid. 

Caustic 
Soda. 

Chloride  of 
Gold  and 
Sodium. 

Nitrate 
of 

Silver. 

Carbolic 

Acid. 

Anthrax  bacillus  .  .  . 

1  •  1100 

1  •  450 

1  :8000 

1  •  20000 

1:300 

Diphtheria  bacillus  
Glanders  bacillus  
Typhoid  bacillus 

1  :700 
1  :200 
1  :300 

-  1  :  300 
1  :  150 
1  :190 

1  :1000 
1:400 
1  :500 

1  :2500 
1  :  4000 
1  :4000 

1  :300 
1  :300 
1  :  200 

Cholera  spirillum  

1  :  1350 

1  :150 

1  :  1000 

1  :4000 

1  :400 

(b)  The  presence  or  absence  of  spores.     The  reproductive  ele- 
ments known  as  spores  have  a  far  greater  resisting  power  to  chemi- 
cal agents,  as  well  as  to  heat,  than  have  the  vegetative  cells.     In 
1  Quoted  from  article  on  "  Germicides  and  Disinfectants/'  in  "  Bacteria,"  p.  212, 


1(;(;  ANTISEPTICS  AND   DISINFECTANTS. 

practical  disinfection,  therefore,  it  is  important  to  know  what  disease 
germs  form  spores  and  what  do  not.  The  following  are  known  to 
form  spores :  The  bacillus  of  anthrax,  the  bacillus  of  tetanus,  the 
bacillus  of  malignant  oedema,  the  bacillus  of  symptomatic  anthrax, 
the  bacillus  of  foul  brood  (infectious  disease  of  bees).  The  following, 
so  far  as  is  known,  do  not  form  spores :  The  pus  cocci  (Staphylo- 
coccus  pyogenes  albus,  aureus,  and  citreus,  and  Streptococcus  pyo- 
genes),  the  micrococcus  of  pneumonia,  the  bacillus  of  typhoid  fever, 
the  bacillus  of  glanders,  the  bacillus  of  diphtheria,  the  spirillum  of 
cholera,  the  spirillum  of  relapsing  fever. 

Many  agents  which  kill  the  growing  bacteria  are  incapable  of 
destroying  the  vitality  of  spores,  and  others  only  do  so  in  much 
stronger  solutions  or  after  a  long  exposure  to  their  action. 

(c)  The  number  of  bacteria  to  be  destroyed.     This  is  an  essen- 
tial factor  which  has  often  been  overlooked  by  those  making  experi- 
ments.   To  destroy  the  bacteria  carried  over  to  five  cubic  centimetres 
of  distilled  water  by  means  of  a  platinum  loop,  is  a  very  different 
matter  from  destroying  the  immensely  greater  number  in  five  cubic 
centimetres  of  a  recent  bouillon  culture. 

(d)  The  nature  and  quantity  of  associated  material.     The 
oxidizing  disinfectants,  like  permanganate  of  potash  and  chloride  of 
lime,  not  only  act  upon  the  bacteria,  destroying  them  by  oxidation, 
but  upon  all  organic  matter  with  which  they  come  in  contact,  and  at 
the  same  time  the  disinfecting  agent  is  destroyed  in  the  chemical 
reaction,  which  is  a  quantitative  one.     The  presence,  therefore,  of 
organic  material  in  association  with  the  bacteria  is  an  important 
factor,  and  if  this  is  in  excess  the  disinfectant  may  be  neutralized 
before  the  living  bacteria  are  destroyed.     Other  substances  which 
precipitate  the  disinfecting  agent  in  an  insoluble  form,  or  decompose 
it .  must  of  course  have  the  same  effect.    Thus  the  presence  of  sodium 
chloride  in  a  culture  medium  would  be  an  important  circumstance  if 
nitrate  of  silver  was  the  agent  being  tested,  as  the  insoluble  chloride 
would  be  precipitated.     And  in  the  case  of  mercuric  chloride  and 
certain  other  metallic  salts  the  presence  of  albumin  very  materially 
influences  the  result.     Van  Ermengem  states  that  the  cholera  spiril- 
lum in  bouillon  is  destroyed  in  half  an  hour  by  mercuric  chloride  in 
the  proportion  of  1 : 00,000,  while  in  blood  serum  1 : 800  was  required 
t«.  destroy  it  in  the  same  time. 

(e)  The  time  of  exjxttturv  is  also  an  important  factor.  Some 
agents  act  very  promptly,  while  others  require  a  considerable  time  to 
effect  the  destruction  of  bacteria  exposed  to  their  action.  Thus  a 
solution  "of  chloride  of  lime  containing  0.12  per  cent  destroys  the 
typhoid  bacillus  and  the  cholera  spirillum  in  five  minutes,  and 
tin-  anthrax  bacillus  iu  one  minute  (Nissen).  On  the  other  hand, 


ANTISEPTICS  AND   DISINFECTANTS.  167 

quicklime  (milk  of  lime)  requires  a  contact  of  several  hours  to  in- 
sure the  destruction  of  pathogenic  bacteria. 

(/)  The  temperature  at  which  the  exposure  is  made  has  a 
material  influence  upon  the  result.  This  is  shown  by  the  experi- 
ments of  Henle  and  of  Nocht.  As  a  general  rule  germicidal  activ- 
ity increases  in  direct  proportion  to  the  increase  in  temperature  from 
20°  C.  upward. 

(g)  The  degree  of  dilution  of  the  disinfecting  agent  is  also  a 
matter  of  importance .  This  is  especially  true  of  solutions  of  acids 
and  alkalies.  When  a  silk  thread  to  which  bacteria  are  attached  is 
suspended  in  an  acid  solution  the  essential  point  is  the  degree  of 
acidity,  and  not  the  quantity  of  acid  in  the  entire  solution.  But  if  a 
solution  of  permanganate  of  potash,  or  any  other  active  oxidizing 
agent,  is  used,  the  principal  question  is  not  the  degree  of  dilution,  but 
the  amount  of  the  disinfecting  agent  present  in  the  solution  used.  A 
grain  of  potassium  permanganate  dissolved  in  two  fluidounces  of 
distilled  water  would  probably  kill  just  as  many  bacteria  as  if  it 
were  dissolved  in  half  a  fluidounce,  although  the  time  required  for 
disinfection  might  be  longer. 

From  what  has  been  said  it  is  evident  that  the  simple  statement 
that  a  certain  agent  is  a  germicide  in  a  certain  proportion  has  but 
little  scientific  value,  unless  we  are  made  acquainted  with  the  condi- 
tions under  which  its  germicidal  action  has  been  tested. 


VIII. 

ACTION  OF  GASES  AND  OF  THE  HALOID  ELEMENTS 
UPON  BACTERIA. 

Oxygen. — Free  oxygen  is  essential  for  the  development  of  a  large 
number  of  species  of  bacteria — aerobics  ;  and  it  completely  prevents 
the  growth  of  others — anaerobics.  Many  bacteria,  even  when  freely 
exposed  in  a  desiccated  condition  to  the  action  of  atmospheric  oxygen, 
retain  their  vitality  for  a  long  time.  The  gradual  loss  of  pathogenic 
power  which  Pasteur  has  shown  occurs  in  cultures  of  the  anthrax 
bacillus  and  the  micrococcus  of  fowl  cholera,  is  ascribed  by  him  to 
exposure  to  oxygen,  and  as  proof  of  this  he  states  that  cultures  kept 
in  hermetically  sealed  tubes  do  not  lose  their  virulence  in  the  same 
degree.  But  other  circumstances  may  influence  the  result.  Thus 
some  of  the  products  of  growth  which  accumulate  in  culture  fluids 
have  an  injurious  effect  upon  the  vitality  of  the  bacteria  which  pro- 
duced them,  and  in  time  may  cause  a  complete  destruction  of  vitality. 
In  cultures  exposed  to  the  air  these  products  would  be  in  a  more 
concentrated  solution  from  the  gradual  evaporation  of  the  culture 
liquid.  It  must  also  be  remembered  that  light  in  the  presence  of 
oxygen  is  a  germicidal  agent. 

The  experiments  of  Frankel  show  that  the  aerobic  bacteria  grow 
abundantly  in  the  presence  of  pure  oxygen,  and  some  species  even 
more  so  than  in  ordinary  air.  Micrococcus  prodigiosus,  however, 
appeared  to  be  unfavorably  affected  by  pure  oxygen,  inasmuch  as  it 
did  not  produce  pigment  so  readily  as  when  cultivated  in  ordinary  air. 

Nascent  oxygen  is  a  very  potent  germicidal  agent,  as  will  be  seen 
in  our  account  of  such  oxidizing  disinfectants  as  potassium  perman- 
^anat<«  and  the  hvporhloritc  of  lime. 

Ozone.— It  was  formerly  supposed  that  ozone  would  prove  to  be 
a  most  valuable  agent  for  disinfecting  purposes  ;  but  recent  experi- 
ments show  that  it  is  not  so  active  a  germicide  as  was  anticipated, 
and  that  from  a  practical  point  of  view  it  has  comparatively  little 
value. 

Lukaschewitsch  found  that  one  gramme  in  the  space  of  a  cubic 
metre  failed  to  kill  anthrax  spores  in  twenty-four  hours.  The  cholera 
>j.ii  -ilium  in  amoist  state  was  killed  in  this  time  by  the  same  amount, 
but  fifteen  hours'  exposure  failed  to  destroy  it.  Ozone  for  these  ex- 
periments was  developed  by  means  of  electricity. 


ACTION   OF   GASES   AND   HALOID   ELEMENTS  UPON  BACTERIA.     169 

Wyssokowicz  found  that  the  presence  of  ozone  in  a  culture  me- 
dium restrained  the  development  of  the  anthrax  bacillus,  the  bacillus 
of  typhoid  fever,  and  others  tested,  but  concludes  that  this  is  rather 
due  to  the  oxidation  of  bases  contained  in  the  nutrient  medium  than 
to  a  direct  action  upon  the  pathogenic  bacteria. 

Sonntag,  in  his  carefully  conducted  experiments,  in  which  a  cur- 
rent of  ozonized  air  was  made  to  pass  over  silk  threads  to  which  were 
attached  anthrax  spores,  had  an  entirely  negative  result.  The  an- 
thrax bacillus  from  the  spleen  of  a  mouse,  and  free  from  spores,  was 
then  tested,  also  with  a  negative  result,  even  after  exposure  to  the 
ozonized  air  for  twenty  minutes  at  a  time  on  four  successive  days.  In 
another  experiment  several  test  organisms  (Bacillus  anthracis,  Bacil- 
lus pneumonise  of  Friedlander,  Staphylococcus  pyogenes  aureus, 
Staphylococcus  pyogenes  albus,  Bacillus  murisepticus,  Bacillus 
crassus  sputigenus)  were  exposed  on  silk  threads  for  twenty-four 
hours  in  an  atmosphere  containing  4. 1  milligrammes  of  ozone  to  the 
litre'of  air  (0. 19  volumes  per  cent).  The  result  was  entirely  negative. 
When  the  amount  was  increased  to  13.53  milligrammes  per  litre  the 
anthrax  bacillus  and  Staphylococcus  pyogenes  albus  failed  to  grow 
after  twenty-four  hours'  exposure.  The  conclusion  reached  by  Ms- 
sen,  from  his  own  experiments  and  a  careful  consideration  of  those 
previously  made  by  others,  is  that  ozone  is  of  no  practical  value  as  a 
germicide  in  therapeutics  or  disinfection. 

Hydrogen. — This  gas  has  no  injurious  effect  upon  bacteria,  as  is 
shown  by  the  fact  that  the  anaerobic  and  facultative  anaerobic  species 
grow  readily  in  an  atmosphere  of  pure  hydrogen. 

Hydrogen  peroxide  in  solution  in  water  is  a  valuable  antiseptic 
and  deodorant,  but  its  value  as  a  germicide  has  been  very  much 
overestimated.  Miquel,  in  his  experiments  to  determine  the  anti- 
septic value  of  various  agents,  places  H2O2  third  in  the  list  of  "  sub- 
stances eminently  antiseptic,"  and  states  that  it  prevents  the  develop- 
ment of  the  bacteria  of  putrefaction  in  the  proportion  of  1: 20,000. 

In  the  writer's  experiments  (1885)  a  solution  was  used  which 
contained  at  first  4.8  per  cent  of  H2O2,  and  five  per  cent  of  sulphuric 
acid  which  was  added  by  the  chemist  who  prepared  the  solution,  to 
prevent  loss  of  the  hydrogen  peroxide.  At  the  end  of  a  month  the 
amount  of  H20a  was  again  estimated,  and  found  to  be  3.98  per  cent. 
Five  weeks  later  the  proportion  was  2.4  per  cent.  Tested  upon 
"broken-down"  beef  tea,  this  solution  was  found  to  destroy  the 
vitality  of  the  bacteria  of  putrefaction  contained  in  it,  in  two  hours' 
time,  in  the  proportion  of  thirty  per  cent  (about  1.2  per  cent  of  H2O2). 
Anthrax  spores  were  killed  in  the  same  time  by  a  twenty-per-cent 
solution  (0.8  per  cent  H2O,).  Tested  upon  a  pure  culture  of  pus 
cocci,  it  was  active  in  the  proportion  of  ten  per  cent  (0. 4  per  cent  of 


170  ACTION  OF   GASES  AND   OF   THE 

H,O,);  a  solution  containing  0.24  per  cent  of  HaOa  failed  to  kill  pus 
cocci.  But  the  solution  used  in  these  experiments  contained  also  five 
per  cent  of  sulphuric  acid,  which  by  itself  kills  micrococci  in  the  pro- 
portion of  1 : 200.  My  conclusion  was  that,  unless  the  chemists  can 
furnish  more  concentrated  solutions  which  will  keep  better  than  that 
with  which  I  experimented,  we  are  not  likely  to  derive  any  practical 
benefit  from  the  use  of  hydrogen  peroxide  as  a  disinfectant. 

Altehof er  more  recently  has  experimented  with  a  solution  contain- 
ing 9.7  per  cent  of  HaOa,  and  reports  the  following  results:  He  added 
to  ninety-eight  cubic  centimetres  of  hydrant  water  two  cubic  centi- 
metres of  a  bouillon  culture  of  the  typhoid  bacillus,  and  to  this  was 
added  sufficient  of  his  aqueous  solution  of  H2O.,  to  make  the  propor- 
tion present  1: 1,000.  At  the  end  of  twenty-four  hours  the  bacillus 
was  proved  by  culture  experiments  to  be  killed.  Water  containing 
the  cholera  spirillum,  treated  in  the  same  way,  was  not  entirely  steril- 
ized, as  a  few  colonies  developed  in  Esmarch  roll  tubes  ;  but  the  gen- 
eral result  of  his  experiments  was  that  the  ordinary  water  bacteVia, 
and  the  pathogenic  bacteria  named  (cholera,  typhoid)  when  sus- 
pended in  water,  required  for  their  destruction  exposure  for  twenty- 
four  hours  in  a  solution  containing  one  part  of  HaO2  in  one  thousand 
of  water. 

Carbon  Dioxide. — The  experiments  of  Frankel  show  that  certain 
bacteria  grow  in  an  atmosphere  of  CO.,  as  well  as  in  the  air  ;  among 
these  are  the  bacillus  of  typhoid  fever  and  the  pneumonia  bacillus 
of  Friedlander.  Other  species  are  slightly  restricted  in  their  growth, 
e.g.  Bacillus  prodigiosus,  Proteus  vulgaris.  Still  others  grow  only 
when  the  temperature  is  elevated,  including  the  pus  cocci  and  the 
bacillus  of  swine  pest.  Most  of  the  saprophytic  bacteria  failed  to 
grow  in  an  atmosphere  of  CO,,  although  their  vitality  was  not  de- 
stroyed by  it.  Certain  pathogenic  species  were,  however,  killed  by 
the  action  of  this  gas,  among  others  the  cholera  spirillum,  Bacillus 
anthracis,  and  Staphylococcus  pyogenes  aureus. 

Leone  and  Hochstetter  had  previously  reported  that  certain  bac- 
teria are  injuriously  affected  by  COa.  Frankel  also  found  that  the 
growth  of  strictly  anaerobic  species  was  restricted  in  an  atmosphere 
of  carbon  dioxide.  The  aerobic  species  which  failed  to  grow  in  pure 
CO,  grew  al.nml.-iiitly  when  a  little  atmospheric  oxygen  was  ad- 
mit i  •••  I .  In  the  experiments  of  Frankla  ad  the  cholera  spirillum  and 
the  Finkler-Prior  spirillum  failed  to  develop  in  an  atmosphere  of 
CO,,  and  at  the  end  of  eight  days  were  no  longer  capable  of  growth 
wh.-n  the  carbon  dioxide  was  replaced  with  atmospheric  air. 

<  Carbonic  Oxide. — Frankland's  experiments  show  that  an  atmo- 
sphere of  this  gas  is  not  favorable  to  the  growth  of  the  cholera  spiril- 
lum or  of  the  Finkl.T-Prior  spirillum,  although  it  did  not  entirely 


HALOID   ELEMENTS   UPON   BACTERIA.  171 

prevent  development,  and  after  seven  days' exposure  the  spirilla  were 
not  all  killed,  although  a  comparatively  small  number  of  colonies  de- 
veloped. Bacillus  pyocyaneus  failed  to  grow  in  an  atmosphere  of 
CO,  but  when  air  was  admitted,  at  the  end  of  seven  or  eight  days, 
abundant  development  occurred. 

Methane,  CH4. — We  have  no  exact  experiments  to  determine 
the  action  of  marsh  gas  in  a  pure  state  on  bacteria,  but  the  experi- 
ments of  Kladakis  upon  illuminating  gas  maybe  taken  as  repre- 
senting approximately  what  might  be  expected  from  exposure  in 
pure  CH4.  An  analysis  of  the  gas  used  in  his  experiments  showed 
it  to  contain  37.97  per  cent  of  hydrogen,  39.37  per  cent  of  methane 
(CH4),  9.99  per  cent  of  nitrogen,  4.29  per  cent  of  ethene  (CaH4),  3.97 
per  cent  of  carbonic  oxide  (CO),  0.61  per  cent  of  oxygen,  and  0.41  per 
cent  of  carbon  dioxide.  As  hydrogen  and  nitrogen  are  neutral,  and 
carbonic  oxide  is  shown  by  the  experiments  of  Frankland  not  to  act 
as  a  germicide  after  several  days"  exposure  to  its  action,  the  positive 
results  obtained  in  the  experiments  of  Kladakis  may  be  ascribed  to 
the  presence  of  CH4  (39.37  per  cent)  or  of  C2H4  (4.29  per  cent),  or  of 
both  together. 

A  large  number  of  microorganisms  were  tested,  and  among  these 
Proteus  vulgaris  alone  grew  in  an  atmosphere  of  illuminating  gas. 
The  others  not  only  failed  to  grow  in  such  an  atmosphere,  but  were 
destroyed  by  it.  Cultures  of  Bacillus  anthracis,  Staphylococcus  pyo- 
genes  aureus,  and  Spirillum  cholera?  Asiatics  were  sterilized  in  half 
an  hour  by  the  action  of  this  gas.  The  gas  was  also  found  to  be  un- 
suitable for  anaerobic  cultures. 

Nitrous  Oxide,  N2O. — The  experiments  of  Frankland,  made 
upon  the  cholera  spirillum,  the  spirillum  of  Finkler-Prior,  and  the 
bacillus  of  green  pus,  gave  results  similar  to  those  obtained  with  CO, 
viz. ,  seven  days'  exposure  in  an  atmosphere  of  this  gas  failed  to  de- 
stroy the  test  organisms,  but  completely  restrained  the  growth  of 
Bacillus  pyocyaneus  and  interfered  materially  with  the  development 
of  the  two  species  of  spirillum  without  entirely  preventing  it. 

Nitrogen  Dioxide,  NO. — Frankland  found  that  his  test  organ- 
isms were  quickly  killed  by  this  gas  (Bacillus  pyocyaneus,  Spirillum 
cholera  Asiatics,  Spirillum  Finkler-Prior). 

Hydrosulphuric  Acid,  H2S. — In  the  experiments  of  Frankland 
this  gas  proved  to  be  quickly  fatal  to  the  bacteria  tested  (Bacillus 
pyocyaneus,  Spirillum  cholera?  Asiatics,  Spirillum  Finkler-Prior). 
On  the  other  hand,  Grauer  found  that  this  gas  did  not  exercise  any 
injurious  influence  upon  the  tubercle  bacillus,  the  bacillus  of  anthrax, 
the  typhoid  bacillus,  or  the  cholera  spirillum,  after  the  exposure  of 
these  microorganisms  in  a  current  of  the  gas  for  an  hour. 

It  has  been  shown  by  the  experiments  of   Holschewnikoff    and 


UTInN    OF   GASES   AND   OF   THE 

others  that  certain  species  of  bacteria  cause  an  abundant  evolution 
of  H,S  as  a  result  of  their  development  in  an  albuminous  medium 
(Bacillus  sulfureus  and  Proteus  sulfureus). 

Sulphur  Dioxide,  SO,.— Very  numerous  experiments  have  been 
made  with  this  gas,  owing  to  the  fact  that  it  has  been  extensively 
used  in  various  parts  of  the  world  for  the  disinfection  of  hospitals, 
ships,  apartments,  clothing,  etc. 

In  the  writer's  experiments,  made  in  1880,  dry  vaccine  virus  on 
ivory  points  was  disinfected  by  exposure  for  twelve  hours  in  an  at- 
mosphere containing  one  volume  per  cent  of  this  gas,  and  liquid 
virus,  exposed  in  a  watch  glass,  by  one-third  of  this  amount.  Sub- 
sequent experiments  (1885)  showed  that  pus  micrococci  were  killed 
by  exposure  for  eighteen  hours  in  a  dry  atmosphere  containing  twenty 
volumes  per  cent  of  S0a,  but  that  four  volumes  per  cent  failed.  In 
the  presence  of  moisture  this  gas  has  considerably  greater  germicidal 
power  than  this,  owing,  no  doubt,  to  the  formation  of  the  more  ac- 
tive agent,  sulphurous  acid  (H,S03).  But  in  a  pure  state  anhydrous 
sulphur  dioxide  does  not  destroy  spores.  The  writer  has  shown  that 
the  spores  of  Bacillus  anthracis  and  Bacillus  subtilis  are  not  killed  by 
contact  for  some  time  with  liquid  SO2  (liquefied  by  pressure).  Koch 
exposed  various  species  of  spore-bearing  bacilli  in  a  disinfection  cham- 
ber for  ninety-six  hours,  the  amount  of  SOa  at  the  outset  of  the  ex- 
periment being  6.13  volumes  per  cent,  and  at  the  end  3.3  per  cent. 
The  result  was  entirely  negative. 

But  in  the  absence  of  spores  the  anthrax  bacillus,  in  a  moist  con- 
dition, attached  to  silk  threads,  was  destroyed  in  thirty  minutes  in 
an  atmosphere  containing  one  volume  per  cent. 

In  another  of  Koch's  experiments  the  amount  of  SO,  in  the  disin- 
fection chamber  was  at  the  outset  0.84  per  cent,  and  at  the  end  of 
twenty-four  hours  0. 55  per  cent.  An  exposure  of  one  hour  in  this  at- 
mosphere killed  anthrax  bacilli  attached  to  silk  threads,  in  a  moist 
condition ;  but  four  hours'  exposure  failed  to  kill  Bacillus  prodigiosus 
growing  on  potato,  while  twenty-four  hours'  exposure  was  successful. 
A  similar  result  was  obtained  with  Bacillus  pyocyaneus. 

Thinot,  as  a  result  of  experiments  made  in  1890,  arrives  at  the 
conclusion  that  the  specific  germs  of  tuberculosis,  glanders,  farcy  of 
cattle,  typhoid  fever,  cholera,  and  diphtheria  are  destroyed  by  twenty- 
four  hours'  exposure  in  an  atmosphere  containing  SO,  developed  by 
the  combustion  of  sixty  grains  of  sulphur  per  cubic  metre.  This 
amount  corresponds  closely  with  that  fixed  by  the  Committee  on  Dis- 
infoctante  of  the  American  Public  Health  Association  on  the  experi- 
mental evidence  obtained  by  the  writer  in  1885.  But  the  committee 
insisted  upon  tin-  pn-s.-nrr  <>f  inuistuiv  and  mado  thetimo  of  exposure 
twelve  hours — "exposure  for  twelve  hours  to  an.  atmosphere  con- 


HALOID   ELEMENTS   UPON   BACTERIA.  173 

taining  at  least  four  volumes  per  cent  of  this  gas  in  the  presence  of 
moisture. " 

Chlorine. — The  haloid  elements  are  active  germicidal  agents, 
especially  chlorine  on  account  of  its  affinity  for  hydrogen,  and  the 
consequent  release  of  nascent  oxygen  when  it  comes  in  contact  with 
microorganisms  in  a  moist  condition.  And  for  the  same  reason  this 
agent  is  a  much  more  active  germicide  in  the  presence  of  moisture 
than  in  a  dry  condition.  The  experiments  of  Fischer  and  Proskauer 
showed  that  when  dried  anthrax  spores  were  exposed  for  an  hour  in 
an  atmosphere  containing  44. 7  per  cent  of  dry  chlorine  they  were  not 
destroyed  ;  but  if  the  spores  were  previously  moistened  and  were  ex- 
posed in  a  moist  atmosphere  for  the  same  time,  four  per  cent  was 
effective,  and  when  the  time  was  extended  to  three  hours  one  per 
cent  destroyed  their  vitality.  The  anthrax  bacillus,  in  the  absence  of 
spores,  was  killed  by  exposure  in  a  moist  atmosphere  containing  1 
part  to  2,500,  the  time  of  exposure  being  twenty-four  hours,  and  the 
same  amount  was  effective  for  Micrococcus  tetragenus  ;  the  strepto- 
coccus of  erysipelas  and  the  micrococcus  of  fowl  cholera  were  killed  in 
three  hours  by  1  : 2,500,  and  in  twenty-four  hours  by  1: 25,000.  The 
bacillus  of  mouse  septicaemia  and  the  tubercle  bacillus  were  killed  in 
one  hour  by  1  : 200. 

In  the  writer's  experiments  (1880)  four  children  were  vaccinated 
with  virus  from  ivory  points  which  had  been  exposed  for  six  hours  in 
an  atmosphere  containing  one-half  per  cent  of  chlorine ;  also  with 
four  points,  from  the  same  lot,  not  disinfected.  Vaccination  was  un- 
successful in  every  case  with  the  disinfected  points,  and  successful 
with  those  not  disinfected.  Koch  found  that  anthrax  spores  failed 
to  grow  after  twenty-four  hours'  exposure  in  chlorine  water.  In 
the  experiments  of  De  la  Croix  to  determine  the  antiseptic  power  of 
this  agent,  it  was  found  that  when  present  in  unboiled  beef  infusion 
in  the  proportion  of  1  : 15,000  no  development  of  bacteria  occurred. 
Miquel  gives  the  antiseptic  value  of  chlorine  as  1  : 4,000. 

Chloroform. — Immersion  for  one  hundred  days  in  chloroform 
does  not  destroy  the  vitality  of  anthrax  spores  (Koch).  This  agent 
is  without  effect  on  the  virus  of  symptomatic  anthrax  (Arloing, 
Cornevin,  and  Thomas).  Salkowski  found  that  the  anthrax  bacillus 
in  the  absence  of  spores,  and  the  cholera  spirillum,  were  killed  by 
being  immersed  in  chloroform  water  for  half  an  hour.  Kirchner 
reports  still  more  favorable  results.  In  his  experiments  a  one-per- 
cent solution  killed  the  cholera  spirillum  in  less  than  a  minute,  and 
a  one-quarter-per-cent  solution  in  an  hour.  But  the  typhoid  bacillus 
required  at  least  one-half  per  cent  acting  for  an  hour. 

Iodine. — In  the  writer's  experiments  (1880)  iodine  in  aqueous 
solution  with  potassium  iodide  was  found  to  be  fatal  to  Micrococcus 


174  ACTION   OP  GASES  AND   OF   THE 

pneumonia*  crouposa^in  the  proportion  of  I  :  1,000,  and  to  the  staphy- 
lococci  of  pus  in  1  :  500— time  of  exposure  two  hours.  Iodine  water 
was  found  by  Koch  to  destroy  the  vitality  of  anthrax  spores  in 
twenty-four  hours,  but  a  two-per-cent  solution  in  alcohol  failed  to 
destroy  anthrax  spores  in  forty-eight  hours.  In  the  experiments  of 
Schill  and  Fischer  twenty  hours'  contact  with  a  solution  of  the 
strength  of  1  :  500  failed  to  destroy  the  virulence  of  tuberculous  spu- 
tum, as  tested  by  inoculation  experiments.  The  antiseptic  value  of 
iodine  is  given  by  Miquel  as  1  : 4,000. 

Bromine.—  Fischer  and  Proskauer  have  studied  the  action  of 
bromine  vapor  upon  various  microorganisms.  They  found  that  ex- 
posure for  three  hours  in  a  dry  atmosphere  to  three  per  cent  does 
not  destroy  the  tubercle  bacillus  in  sputum  or  the  spores  of  an- 
thrax. But  when  the  atmosphere  is  saturated  with  moisture  1  :  500 
is  effective  ;  and  when  the  time  of  exposure  was  extended  to  twenty- 
four  hours,  1  : 3,500.  A  two-per-cent  solution  destroys  the  vitality 
of  anthrax  spores  in  twenty-four  hours  (Koch).  Bromine  vapor  is 
an  active  agent  for  the  destruction  of  the  virus  of  symptomatic  an- 
thrax (Arloing,  Cornevin,  and  Thomas).  Miquel  gives  the  antisep- 
tic value  of  bromine  as  1  : 1,666,  which  is  considerably  below  that  of 
chlorine  and  iodine. 

Iodine  Trichloride. — According  to  Behriiig,  we  possess  in  this 
agent  a  disinfectant  which  possesses  the  potency  of  free  chlorine  and 
iodine  without  having  their  disadvantages.  As  prepared  by  O.  Rie- 
del  it  is  a  yellowish-red  powder  of  penetrating  odor.  It  remains  un- 
changed for  weeks  in  concentrated  aqueous  solution  (five  per  cent). 
A  one-per-cent  solution  destroys  anthrax  spores  suspended  in  water 
almost  instantly,  and  a  0.2-per-cent  solution  within  a  few  minutes. 
Anthrax  spores  in  blood  serum  are  killed  by  a  one-per-cent  solution 
in  forty  minutes  (Behring).  Langenbuch  found  that  a  solution  of 
1  : 1 ,000  kills  spores  in  a  short  time,  and  that  when  added  to  nutri- 
ent gelatin  in  the  proportion  of  1  : 1,200  it  restrains  the  develop- 
ment of  bacteria. 

lodoform. — Numerous  experiments  have  been  made  with  this 
agent,  which  slm\v  that  it  has  little,  if  any,  germicidal  power  ;  but 
it  acts  to  some  extent  as  an  antiseptic.  Tilanus  reports  that  the  tu- 
bercle bacillus  will  not  grow  in  glycerin-agar  cultures  to  which  a 
small  quantity  of  iodoform  has  been  added,  and  that  a  pure  culture 
of  the  tubercle  bacillus  was  not  killed  in  six  days  by  exposure  to 
iodoform  vapor,  but  that  after  six  weeks*  exposure  it  failed  to  grow. 
The  experiments  of  Neisser  and  of  Buchner  show  that  while  most 
bacteria  are  not  injuriously  affected  by  exposure  to  iodoform  vapor, 
the  cholera  spirillum  and  the  Finkler-Prior  spirillum  are  restrained  in 
their  growth  by  such  exposure.  When  plate  cultures  of  the  cholera 


HALOID   ELEMENTS   UPON   BACTERIA.  175 

spirillum  were  placed  under  a  bell  jar  beside  iodoform  powder 
no  development  occurred,  but  when  they  were  removed  colonies  de- 
veloped, showing  that  the  spirilla  were  not  killed. 

Iodoform  Ether,  according  to  Yersin,  is  fatal  to  the  tubercle  ba- 
cillus in  one-per-cent  solution  in  five  minutes.  Cadeac  and  Meunier 
found  that  a  saturated  solution  required  thirty-six  hours  to  kill  the 
bacillus  of  typhoid  fever. 

lodol. — In  experiments  made  by  the  writer  (1885)  this  agent  was 
found  to  be  without  germicidal  power.  Riedlin  found  it  without  any 
action,  even  upon  the  cholera  spirillum. 

Hydrofluoric  Acid,  HF1. — From  a  series  of  experiments  made 
with  this  gas,  Grancher  and  Chautard  arrive  at  the  conclusion  that 
"  the  direct  and  prolonged  action  of  hydrofluoric  acid  upon  the  tuber- 
cle bacillus  diminishes  its  virulence  but  does  not  kill  it." 

Sozoiodol  ^4ctd,according  to  Draer,is  a  phenol,  in  which  two  atoms 
of  hydrogen  are  replaced  by  two  of  iodine  and  one  atom  by  the  group 
HSO3.  This  acid  and  its  salts  with  soda,  potash,  zinc,  and  mercury 
have  been  tested  by  the  author  named.  The  acid  and  its  salt  with 
mercury  were  found  to  destroy  the  cholera  spirillum  in  two  hours'  time 
in  two-per-cent  solution.  A  two-per-cent  solution  of  phenol  would 
have  accomplished  the  same  result  and  in  less  time.  Tribromphenol, 
according  to  Draer,  is  less  active  than  sozoiodol  acid ;  and  it  appears 
from  the  experimental  evidence  on  record  that  combinations  of 
iodine,  chlorine,  or  bromine  with  phenol  are  less  active  that  the 
haloid  elements  alone.  According  to  Karpow  (1893)  monochlor- 
phenol,  tested  upon  anthrax  spores  attached  to  silk  threads,  proved 
to  be  decidedly  more  active  than  phenol. 

Nosophen  (tetraiodphenolphthalein),  according  to  Li  even  (1895) 
contains  sixty-one  per  cent  of  iodine.  It  is  entirely  insoluble  in 
water.  When  added  to  nutrient  gelatin  in  the  proportion  of  one- 
quarter  per  cent  it  prevented  the  development  of  the  anthrax  bacillus 
and  of  Staphylococcus  aureus,  but  failed  to  prevent  the  development 
of  Bacillus  pyocyaneus  (Lieven). 


IX. 
ACTION  OF  ACIDS  AND  ALKALIES. 

Sulphuric  Acid,  H,SO4.— The  experiments  of  Koch  (1881) 
showed  that  anthrax  spores  were  still  capable  of  growing  after  ex- 
posure in  a  one-per-cent  solution  of  sulphuric  acid  for  twenty  days. 
In  the  writer's  experiments  (1885)  a  four-per-cent  solution  failed  to 
destroy  the  spores  of  Bacillus  subtilis  in  four  hours,  and  an  eight- 
per-cent  solution  was  found  to  be  required  for  the  sterilization  of 
culture  fluids  containing  spores  ;  but  the  multiplication  of  the  bacte- 
ria of  putrefaction  was  prevented  by  the  presence  of  this  acid  in  a 
culture  solution  in  the  proportion  of  1  :  800.  Pus  micrococci  were 
destroyed  by  exposure  for  two  hours  in  a  solution  containing  1  :  200. 

The  experiments  of  Boer  show  that  there  is  a  considerable  differ- 
ence in  the  resisting  power  of  different  pathogenic  bacteria.  The 
time  of  exposure  being  two  hours,  cultures  in  bouillon  twenty-four 
hours  old  gave  the  following  results  : 


Restrains 
development. 

Destroys 
vitality. 

1  •  2550 

1  :  1300 

Diphtheria  bacillus  

1  :  2050 

1  :500 

Glanders  bacillus  

1  :  750 

1  :  200 

Typhoid  bacillus  

1  :  1550 

1  :500 

Cholera  spirillum  

1  :7000 

1  :1300 

Leitz,  in  his  studies  relating  to  the  bacillus  of  typhoid  fever, 
reports  the  following  results :  The  dejections  of  typhoid  patients, 
i nixed  with  an  equal  proportion  of  the  disinfecting  solution,  were 
.sterilized  by  a  five-per-cent  solution  of  sulphuric  acid  in  three  days. 
A  pure  culture  was  sterilized  in  fifteen  minutes  by  two  per  cent,  and 
in  liv»>  minutes  by  five  per  cent. 

Sulphurous  Acid,  H.SO,.— In  the  writer's  experiments  (1885) 
micrococci  were  destroyed  in  two  hours  by  1  :  2,000  by  weight  of  SO, 
added  to  water.  Kitasato  found  that  solutions  of  sulphurous  acid 
m  the  proportion  of  0.28  per  cent  killed  the  typhoid  bacillus,  and 
<>.  1 48  per  cent  the  cholera  spirillum.  De  la  Croix  found  that  one 


ACTION   OF  ACIDS   AND   ALKALIES.  177 

gramme  of  SO2  added  to  two  thousand  of  bouillon  prevents  the  de- 
velopment of  putrefactive  bacteria  and  after  a  time  destroys  the 
vitality  of  these  bacteria.  The  writer  found  that  pus  cocci  failed  to 
grow  in  a  culture  solution  containing  one  part  of  SO2  in  five  thousand 
of  water. 

Nitric  Acid,  HNO3. — In  the  writer's  experiments  an  eight-per- 
cent solution  which  contained  0.819  gramme  of  HNO3  in  each  cubic 
centimetre  sterilized  broken-down  beef  tea  containing  spores,  and 
five  per  cent  failed  to  do  so.  Kitasato,  in  experiments  upon  the  chol- 
era spirillum  and  typhoid  bacillus,  obtained  results  corresponding 
with  those  obtained  with  hydrochloric  acid — 0. 2  per  cent  destroyed 
vitality  at  the  end  of  four  or  five  hours.  In  these  experiments  the 
acid  used  contained  0.35  gramme  HNO3  in  one  cubic  centimetre. 

Nitrous  Acid. — In  the  writer's  experiments  on  vaccine  virus  (1880) 
exposure  for  six  hours  in  an  atmosphere  containing  one  per  cent  of 
nitrous  acid  destroyed  the  virulence  of  dried  virus  upon  ivory  points. 

Hydrochloric  Acid,  HC1. — Anthrax  spores  are  destroyed  in  ten 
days  by  a  two-per-cent  solution,  but  not  in  five  days  (Koch).  Tested 
upon  broken-down  beef  tea  containing  spores  of  Bacillus  subtilis,  it 
was  effective  in  two  hours  in  the  proportion  of  fifteen  per  cent,  but 
failed  in  ten  per  cent  (Sternberg).  In  the  experiments  of  Kitasato  this 
acid  destroyed  the  typhoid  bacillus  in  five  hours  in  the  proportion  of 
0.2  per  cent,  and  the  cholera  spirillum  in  0.132  per  cent — the  acid 
used  contained  0. 26  gramme  HC1  in  one  cubic  centimetre.  We  give 
the  more  recent  determinations  of  Boer  in  tabular  form.  Its  germi- 
cidal  power  was  tested  upon  bouillon  cultures  which  had  been  kept 
for  twenty-four  hours  in  an  incubating  oven ;  time  of  exposure  to 
the  action  of  the  acid  solution,  two  hours. 


Restrains 
development. 

Destroys 
vitality. 

Anthrax  bacillus  

1  :  3400 

1  :  1100 

Diphtheria  bacillus     .  .                       .             ... 

1  :  3400 

1  -700 

Glanders  bacillus  <           

1  :700 

1-200 

Typhoid  bacillus  

1  :  2100 

1  :300 

Cholera  spirillum  

1  :  5500 

1  :  1350 

Chromic  Acid. — In  Koch's  experiments  a  one-per-cent  solution 
destroyed  anthrax  spores  in  from  one  to  two  days.  In  the  propor- 
tion of  1  : 5,000  it  prevents  the  development  of  putrefactive  bacteria 
(Miquel). 

Osmic  Acid. — A  solution  of  one  per  cent  kills  anthrax  spores  in 
twenty-four  hours  (Koch).  It  is  an  antiseptic  in  the  proportion  of 
1  :6,666  (Miquel). 

Phosphoric  Acid. — Exposure  for  four  or  five  hours  to  a  solution 
12 


178  ACTION  OF  ACIDS  AND   ALKALIES. 

containing  0.3  per  cent  destroys  the  typhoid  bacillus,  and  0.183  per 
cent  the  cholera  spirillum  (Kitasato).  The  acid  used  contained  0. 1  :>•> 
gramme  H,PO4  in  one  cubic  centimetre. 

.Acetic  Acid.— A  five-per-cent  solution  failed  to  kill  anthrax 
spores  after  five  days'  exposure  (Koch).  In  Abbott's  experiments 
glacial  acetic  acid  in  fifty-per-cent  solution  failed  in  two  hours  to  kill 
anthrax  spores,  but  micrococci  were  killed  by  two  hours'  exposure  to 
a  one-per-cent  solution.  A  solution  of  1  : 300  of  glacial  acetic  acid 
destroys  the  cholera  spirillum  in  half  an  hour  (Van  Ermengem).  In 
the  proportion  of  0.25  per  cent  it  restrains  the  growth  of  the  typhoid 
bacillus,  and  0.3  per  cent  destroys  its  vitality  after  five  hours'  expo- 
sure ;  the  cholera  spirillum  fails  to  grow  in  presence  of  0.132  per  cent 
and  is  destroyed  by  0.2  per  cent  (Kitasato). 

Lactic  Acid. — The  bacillus  of  typhoid  fever  is  killed  in  five  hours 
by  a  solution  containing  0.4  per  cent,  the  cholera  spirillum  by  0.3  per 
cent  (Kitasato). 

Citric  Acid. — The  bacillus  of  typhoid  fever  is  killed  in  five  hours 
by  0.43  per  cent,  the  cholera  spirillum  by  0.3  percent  (Kitasato). 
The  cholera  spirillum  is  killed  in  half  an  hour  by  1  : 200  (Van  Er- 
mengem). 

Oxalic  Acid. — The  typhoid  bacillus  requires  a  solution  of  0.36 
per  cent,  the  cholera  spirillum  one  of  0. 28  per  cent,  to  destroy  vitality 
in  five  hours  (Kitasato). 

Boracic  Acid. — In  the  writer's  experiments  (1883)  a  saturated 
solution  failed  to  kill  pus  cocci  in  two  hours.  A  five-per-cent  solu- 
tion failed  to  destroy  anthrax  spores  in  five  days  (Koch).  The 
typhoid  bacillus  is  killed  in  five  hours  by  2.7  percent,  the  cholera 
^jiirillum  by  1.5  per  cent  (Kitasato).  According  to  Arloing,  Corne- 
vin,  and  Thomas,  the  fresh  virus  of  symptomatic  anthrax  requires 
exposure  to  a  twenty-per-cent  solution  for  forty-eight  hours  for  the 
destruction  of  vitality.  Boracic  acid  acts  as  an  antiseptic  in  the  pro- 
portion of  1  : 143  (Miquel). 

Salicylic  Acid. — In  the  writer's  experiments  this  agent  was  dis- 
solved by  the  addition  of  sodium  biborate,  which  by  itself  has  no 
germicidal  power.  A  two-per-cent  solution  was  found  to  destroy  pus 
cocci  in  two  hours.  Dissolved  in  oil  or  in  alcohol  a  five-per-cent  so- 
1  ut ion  does  not  destroy  anthrax  spores  (Koch).  Micrococci  are  de- 
stroyed by  solutions  containing  1  : 400  (Abbott).  The  typhoid  bacillus 
is  killed  in  five  hours  by  1.6  per  cent,  the  cholera  spirillum  by  1.3  per 
cent  (Kitasato).  A  one-per-cent  solution  destroys  Micrococcus  Pas- 
teuri  in  half  an  hour  (Sternberg).  It  is  an  antiseptic  in  the  propor- 
tion of  1  :  1,000  (Miquel).  A  solution  of  2.5  per  cent  kills  the  tubercle 
bacillus  in  six  hours  (Yersin).  In  the  proportion  of  1  : 300it  destroys 
the  cholera  spirillum  in  half  an  hour  (Van  Ermengem). 


ACTION   OF  ACIDS  AND   ALKALIES.  179 

Benzoic  Acid. — According  to  Miquel,  this  acid  restrains  the  de- 
velopment of  putrefactive  bacteria  when  present  in  bouillon  in  the 
proportion  of  1: 909.  In  the  proportion  of  1  :  2,000  it  retards  the  de- 
velopment of  anthrax  spores  (Koch). 

Formic  Acid. — The  typhoid  bacillus  is  restrained  in  its  growth  by 
0.25  per  cent,  and  is  killed  in  five  hours  by  0.35  per  cent,  the  cholera 
spirillum  by  0.22  per  cent  (Kitasato). 

Tannic  Acid. — A  solution  of  one  per  cent  kills  Micrococcus  Pas- 
teuri  in  the  blood  of  a  rabbit  in  half  an  hour  (Sternberg).  A  five- 
per-cent  solution  failed  in  ten  days  to  destroy  anthrax  spores  (Koch). 
A  twenty-per-cent  solution  failed  in  two  hours  to  destroy  the  vitality 
of  spores  of  the  anthrax  bacillus  or  of  Bacillus  subtilis  (Abbott). 
Micrococci  are  destroyed  by  1  : 400,  and  1  : 800  failed  (Abbott).  A 
twenty-per-cent  solution  has  no  effect  upon  the  virus  of  symptomatic 
anthrax  (Arloing,  Cornevin,  and  Thomas).  A  solution  of  1.66  per 
cent  kills  the  typhoid  bacillus  in  five  hours,  and  1.5  per  cent  the 
cholera  bacillus  in  the  same  time  (Kitasato).  It  restrains  the  devel- 
opment of  putrefactive  bacteria  in  the  proportion  of  1  :  207  (Miquel). 

Tartaric  Acid. — A  twenty-per-cent  solution  of  this  acid  fails, 
after  two  hours'  exposure,  to  destroy  the  spores  of  Bacillus  anthracis 
or  Bacillus  subtilis.  Micrococci  are  killed  by  two  hours'  exposure  in 
a  solution  containing  1  :  400  (Abbott). 

Malic  Acid. — This  was  found  by  Kitasato  to  correspond  with 
citric  acid  in  its  germicidal  power. 

Valerianic  Acid. — A  five-per-cent  solution  in  ether  failed  in  five 
days  to  destroy  anthrax  spores  (Koch). 

Oleic  Acid. — A  solution  of  five  percent  in  ether  does  not  destroy 
anthrax  spores  in  five  days  (Koch). 

Thymic  Acid. — In  the  proportion  of  1  : 500  this  acid  prevents  the 
putrefactive  decomposition  of  beef  tea  (Miquel). 

Butyric  Acid. — Five  days'  immersion  in  this  acid  failed  to  de- 
stroy anthrax  spores  (Koch). 

Arsenious  Acid. — A  one-per-cent  solution  destroys  the  vitality 
of  anthrax  spores  in  ten  days,  but  failed  to  do  so  in  six  days  (Koch). 
In  the  proportion  of  1  : 166  it  prevents  putrefactive  changes  in  bouillon 
(Miquel). 

Gallic  Acid. — Abbott  found  this  acid  to  destroy  the  bacteria  in 
broken-down  beef  tea  in  the  proportion  of  2.37  per  cent,  but  it  failed 
to  destroy  anthrax  spores  in  two  hours  in  the  same  proportion.  Mi- 
crococci were  killed  in  two  hours  by  1  : 142,  while  1  :  250  failed. 

ALKALIES. 

Potassium  Hydroxide,  KHO. — In  the  writer's  experiments  aten- 
per-cent  solution  of  caustic  potash  was  fatal  to  pus  cocci,  and  an 


ACTION   OF  ACIDS  AND   ALKALI Ks. 

eight-per-cent  solution  failed — two  hours'  exposure.  Exposure  for 
twenty-four  hours  to  a  ten-per-cent  solution  failed  to  kill  the  tubercle 
bacillus  (Schill  and  Fischer).  A  solution  of  one  per  cent  kills  the 
anthrax  bacillus,  the  bacillus  of  rothlauf,  and  several  others  (Jager). 
The  addition  of  0. 14  per  cent  restrains  the  development  of  the  typhoid 
Iwcillus,  and  0.18  per  cent  kills  this  bacillus  in  four  or  five  hours;  the 
cholera  spirillum  failed  to  grow  in  cultures  containing  0. 18  per  cent 
and  was  killed  by  0.237  per  cent  in  the  same  time  (Kitasato). 

Sodium  Hydroxide,  NaHO.— The  experiments  of  Jager  and  of 
Kitasato  show  that  soda  has  about  the  same  germicidal  power  as 
caustic  potash.  Boer  obtained  the  following  results  with  bouillon 
cultures  after  two  hours'  exposure:  Anthrax  bacillus,  1  :  450  ;  diph- 
theria bacillus,  1  : 300  ;  glanders  bacillus,  1  : 150  ;  typhoid  bacillus, 
1  : 100  ;  cholera  spirillum,  1  : 150.  In  about  one-half  the  amount 
required  to  destroy  vitality  the  development  of  the  above-named  bac- 
teria was  prevented.  In  the  proportion  of  1  :  56  it  acts  as  an  anti- 
septic (Miquel). 

Ammonia,  NH3. — In  Kitasato 's  experiments  the  typhoid  bacillus 
wan  destroyed  in  five  hours  by  0. 3  per  cent  of  NH3,  and  the  cholera 
spirillum  by  about  the  same  amount.  Boer  obtained  the  following 
results,  the  time  of  exposure  being  two  hours  :  Anthrax  bacillus. 
1 :  :HX) :  diphtheria  bacillus,  1  :  250  ;  glanders  bacillus,  1 : 250  ;  typhoid 
I>acillu8,  1 : 200 ;  cholera  spirillum,  1  : 350.  The  growth  of  the  an- 
thrax bacillus  and  of  the  diphtheria  bacillus  in  culture  solutions  was 
prevented  by  1  :  050. 

Calcium  Hydroxide,  Ca2HO. — According  to  Kitasato,  the  ty- 
phoid bacillus  and  the  cholera  spirillum,  in  bouillon  cultures,  are 
killed  in  four  or  five  hours  by  the  addition  of  0.1  per  cent  of  calcium 
•  »\  i<te.  Liborius  had  previously  reported  still  more  favorable  results, 
l>i it  his  bouillon  cultures  were  largely  diluted  with  distilled  water. 

From  a  practical  point  of  view  the  experiments  of  Pfuhl  are  more 
valuable.  Calcium  hydrate  was  added  to  the  dejections  of  typhoid 
jwitieiits.  When  added  in  the  proportion  of  three  per  cent  steriliza- 
tinii  was  effected  in  six  hours,  and  by  six  per  cent  in  two  hours. 

When  milk  of  lime  containing  twenty  per  cent  of  calcium  hydrate 
w;w  used  the  results  were  still  more  favorable,  the  typhoid  bacillus 
and  cholera  spirillum  being  killed  in  one  hour  by  the  addition  of 
two  per  cent  of  the  disinfectant.  The  practical  value  of  lime- wash 

<I>H'"<1  to  walls  has  been  determined  by  Jager.  Silk  threads  soaked 
in  cultures  of  various  pathogenic  bacteria  were  attached  to  boards 
.u  i.l  th<>  li mo-wash  applied  with  a  camel's-hair  brush.  Anthrax  ba- 
nlh  (without  spoivs),  th<>  glanders  bacillus,  Staphylococcus  pyogene? 

nnvus.  and  several  other  pathogenic  bacteria  were  killed  by  a  single 
application  after  twenty-four  hours,  but  the  tubercle  bacillus  was  not 


ACTION   OF   ACIDS   AND    ALKALIES.  181 

killed  by  three  successive  applications.  Iii  the  writer's  experiments 
(1885)  the  typhoid  bacillus  and  Staphylococcus  pyogenes  aureus  were 
killed  in  two  hours  by  a  solution  containing  1 : 40  of  calcium  oxide, 
and  1 :  80  failed.  Spores  of  the  anthrax  bacillus  and  of  several  other 
spore-forming  species  were  not  killed  by  two  hours'  exposure  to  a 
milk  of  lime  containing  twenty  per  cent  of  calcium  oxide. 

Potash  Soap  has  been  shown  by  Jolles  (1895)  to  have  considerable 
germicidal  value.  In  experiments  with  a  soap  containing  67.44 
per  cent  of  fat  acids,  10.4  per  cent  of  combined  alkali,  and  0.041 
per  cent  of  free  alkali,  the  following  results  were  obtained:  The 
typhoid  bacillus  was  destroyed  at  18°  C.  by  a  one-per-cent  solution 
in  twenty-four  hours;  by  a  six-per-cent  solution  in  thirty  minutes. 
The  Bacillus  coli  communis  required  somewhat  stronger  solutions  or 
longer  exposure — eight-per-cent  solution  required  thirty  minutes. 
These  experiments  show  that  scrubbing  with  soap  and  water  is  a 
reliable  method  of  disinfecting  surfaces.  Solutions  of  potash — com- 
mon lye — or  of  soda  also  are  useful  for  certain  purposes  in  domes- 
tic disinfection,  and  scientific  researches  justify  the  continued  use  of 
the  cleansing  methods  which  have  heretofore  been  in  use  by  careful 
housewives. 


X. 
ACTION  OF  SALTS. 

WHILE  some  of  the  metallic  salts,  and  especially  those  of  mer- 
cury, silver,  and  gold,  have  remarkable  germicidal  power,  others, 
even  in  concentrated  solutions,  do  not  destroy  the  vitality  of  bacteria 
exposed  to  their  action.  For  convenience  of  reference  we  shall  con- 
sider the  agents  in  this  group  in  alphabetical  order,  but  first  we  give 
Miquel's  tables  of  antiseptic  value.  This  author  recognizes  the  im- 
portance of  experiments  to  determine  the  restraining  power  of  chem- 
ical agents  for  various  species  of  pathogenic  bacteria,  but  says  :  "  As 
to  me,  faithful  to  a  plan  I  adopted  at  the  outset,  I  will  treat  the  sub- 
ject in  a  more  general  manner  by  making  known  simply  the  mini- 
mum weight  of  the  substances  capable  of  preventing  the  evolution  of 
any  bacteria  or  germs.  The  method  adopted  is  very  simple.  To  a 
liquid  always  comparable  to  itself  it  is  sufficient  at  first  to  add  a 
known  weight  of  the  antiseptic  and  some  atmospheric  germs  or  adult 
bacteria,  and  to  vary  the  quantity  of  the  antiseptic  until  the  amount 
is  ascertained  which  will  preserve  indefinitely  the  liquid  from  putre- 
faction. In  order  to  obtain  germs  of  all  kinds  in  a  dry  state  it  suf- 
fices to  take  them,  where  they  are  most  abundant,  in  the  dust  col- 
lected in  the  interior  of  houses  or  of  hospitals;  and  to  procure  a 
\  triety  of  adult  bacteria  we  may  take  the  water  of  sewers." 


SUBSTANCES  EMINENTLY  ANTISEPTIC. 


Efficient  in  the 
proportion  of — 


Mercuric  iodide,          .  .  .  .  .  .  1 : 40000 

Silver  iodide,         ......  1:33000 

Hydrogen  peroxide,  .  .  .  .  .  .  1 : 20000 

Mercuric  chlorid*  .....  l:143lO 

Silver  nitrate,  .  .  .  .  1 : 12500 

SUBSTANCES  VERY  STRONGLY   ANTISEPTIC. 

Osmicacid,  .  .  i:6666 

(Jnromio  acid.  .....  l-5iiOO 

Chlorine,  1:'4000 

iodine,  .....  1:4000 

Ohloride  of  gold, .  .....  l:40uO 

Bichloride  of  platinum.         .....  1:3333 

Hydrocyanic  acid.  .  1:2500 


ACTION   OF   SALTS.  183 

Bromine,          .......  1:1666 

Cupric  chloride,    .  .  .  .  .  .  1 : 1428 

Thymol, 1:1340 

Cupric  sulphate,  .  .  .  .  .  1:1111 

Salicylic  acid,  .  .  .  .  .  .  1 : 1000 


SUBSTANCES  STRONGLY  ANTISEPTIC. 

Benzoic  acid,         .  .  .  .  .  .  1 : 909 

Potassium  bichromate,  .  .  .  .  .       1 : 909 

Potassium  cyanide,  .  .  .  .  .  1 : 909 

Aluminum  chloride,  .  .  .  .  .       1 : 714 

Ammonia,  ......  1:714 

Zinc  chloride,  .  .  .  .  .  1 : 526 

Mineral  acids,        .  .  .  .  .          1:500  to  1:333 

Thymicacid,  .  .  .  .  .  .  .1:500 

Lead  chloride,       .  .  .  .  .  .  1:500 

Nitrate  of  cobalt,         .  .  .  .  .  .1:476 

Sulphate  of  nickel,  .  .  .  .  .  1 : 400 

Nitrate  of  uranium,    ......       1:356 

Carbolic  acid,        .  .  .  .  .  .  1:333 

Potassium  permanganate,     .  .  .  .  .       1 : 285 

Lead  nitrate,         .  .  .  .  .  .  1 : 277 

Alum, 1:222 

Tannin, 1:207 


SUBSTANCES  MODERATELY   ANTISEPTIC. 

Bromhydrate  of  quinine,       .....       1:182 

Arsenious  acid,     .             .            .            .            .            .  1:166 

Boracic  acid,   .            .            .            .            .            .  1 : 143 

Sulphate  of  strychnia,     .            .            .            .            .  1 : 143 

Arsenite  of  soda,         .             .            .            .            .  .1:111 

Hydrate  of  chloral, 1:107 

Salicylate  of  soda,      .            .            .            .            .  .1:100 

Ferrous  sulphate,              .            .            .            .            .  1 : 90 

Caustic  soda,  .             .            .            .            .            .  1 : 56 


SUBSTANCES   FREELY  ANTISEPTIC. 

Perchloride  of  manganese,          .            .            .  .             1 : 40 

Calcium  chloride,       .             .            .            .            .  1 : 25 

Sodium  borate,      .             .             .            .            .  .             1 : 14 

Muriate  of  morphia,  .             .            .            .             .  .       1 : 13 

Strontium  chloride,          .             .            .            .  .             1 : 12 

Lithium  chloride,        .            .            .            .            .  .1:11 

Barium  chloride,  .             .            .            .            .  .             1 : 10 

Alcohol 1:10 


SUBSTANCES   VERY   FEEBLY   ANTISEPTIC. 

Ammonium  chloride,  .            .            .            .            .             1:9 

Potassium  arsenite,    .  .            .            .            .            .1:8 

Potassium  iodide,  .            .            .            .            .             1:7 

Sodium  chloride,         .  .            .            .            .             .1:6 

Glycerin  (sp.  gr.  1.25),  .... 

Ammonium  sulphate,  .            .            .            .            .1:4 

Sodium  hyposulphite,  .            .            .            .            .             1:3 


}g4  ACTION   OF   SALTS. 

ANTISEPTIC!   AND   GERMICIDAL   VALUE   OF   VARIOUS   SALTS, 
ARRANGED   ALPHABETICALLY. 

A lum.—  Antiseptic  in  the  proportion  of  1  :  222  (Miquel). 

Aluminum  Acetate. — According  to  De  la  Croix,  this  salt  is  an 
antiseptic  in  the  proportion  of  1  :  6,310.  Kuhn  found  it  to  be  anti- 
septic in  1  :5,250. 

Aluminum  Chloride. — Antiseptic   in   the  proportion  of   1  : 714 

(Miquel). 

Ammonium  Carbonate. — When  present  in  the  proportion  of 
1  : 125  it  restrains  the  development  of  typhoid  bacilli,  and  in  five 
hours'  time  it  kills  these  bacilli  in  the  proportion  of  1  : 100 ;  the 
cholera  spirillum  is  killed  in  the  same  time  by  1  :  77  (Kitasato). 

Ammonium  Chloride. — Antiseptic  in  the  proportion  of  1:9 
(Miquel).  A  five-per-cent  solution  does  not  kill  anthrax  spores  in 
twenty-five  days  (Koch). 

Ammonium  Fluosilicate. — The  bacillus  of  anthrax  and  of  ty- 
phoid fever  fail  to  grow  in  nutrient  gelatin  containing  1  : 1,000,  and 
a  two-per-cent  solution  kills  anthrax  spores  in  one-quarter  to  three- 
quarters  of  an  hour  (Faktor). 

Ammonium  Sulphate. — Antiseptic  in  the  proportion  of  1:4 
(Miquel).  A  five-per-cent  solution  failed  in  two  days  to  kill  an- 
thrax spores,  but  was  effective  in  five  days  (Koch). 

Barium  Chloride  is  an  antiseptic  in  the  proportion  of  1  : 10 
(Miquel). 

Calcium  Chloride  is  an  antiseptic  in  the  proportion  of  1  : 25 
(Miquel).  A  saturated  solution  does  not  destroy  anthrax  spores 
(Koch). 

Calcium  Hypochlorite. — This  is  a  powerful  germicidal  agent 
and  has  great  value  as  a  practical  disinfectant.  Good  chloride  of 
lime  contains  from  twenty-five  to  thirty  per  cent  of  available  chlo- 
rine as  hypochlorite.  The  experiments  made  by  the  Committee  on 
Disinfectants  of  the  American  Public  Health  Association  in  1885 
showed  that  a  solution  containing  0. 25  per  cent  of  chlorine  as  hypo- 
chlorite is  an  effective  germicide,  even  when  allowed  to  act  only 
for  one  or  two  minutes.  In  Bolton's  experiments  a  solution  of  chlo- 
ride of  lime  of  1  : 2,000  (available  chlorine  0.015)  destroyed  the  ty- 
phoid bacillus  and  the  cholera  spirillum  in  two  hours.  For  the  de- 
-  traction  of  anthrax  spores  a  one-per-cent  solution  was  required 
(available  chlorine  0.3  per  cent).  Nissen  found  that  the  typhoid 
bacillus  and  the  cholera  spirillum  are  destroyed  with  certainty  in 
live,  minutes  by  a  solution  containing  0.12  percent,  anthrax  bacilli 
in  one  minute  by  o.l  per  cent,  Staphylococcus  pyogenes  aureus  in 
one  minute  by  0.2  per  cent,  anthrax  spores  in  thirty  minutes  by  a 


ACTION   OF   SALTS.  185 

five-per-ceiit  solution  and  in  seventy  minutes  by  a  one-per-cent  solu- 
tion. Experiments  made  by  the  same  author  upon  the  sterilization 
of  faeces  showed  that  0. 5  per  cent  to  one  per  cent  could  be  relied  .upon 
to  destroy  the  typhoid  bacillus  or  the  cholera  spirillum  in  faeces  in 
ten  minutes. 

Chloral  Hydrate. — Antiseptic  in  the  proportion  of  1  : 107  (Mi- 
quel).  A  tvventy-per-cent  solution  destroys  pus  cocci  in  two  hours 
(Sternberg). 

Cupric  Chloride. — Antiseptic  in  the  proportion  of  1  •  1,428 
(Miquel). 

Cupric  Sulphate. — Antiseptic  in  the  proportion  of  1  :  111  (Mi- 
quel). Kills  the  cholera  spirillum  in  the  proportion  of  1  : 3,000  in 
ten  minutes  (Nicati  and  Rietsch).  Destroys  the  cholera  spirillum  in 
bouillon  cultures  in  less  than  half  an  hour  in  1  :  600,  and  in  four 
hours  in  1  : 1,000  ;  cultures  in  blood  serum  require  1  :  200  (Van  Er- 
mengem).  A  solution  of  1  : 20  kills  the  typhoid  bacillus  in  ten  min- 
utes (Leitz).  This  salt  failed,  in  the  writer's  experiments,  to  kill  the 
spores  of  Bacillus  anthracis  and  Bacillus  subtilis  in  two  hours'  time 
in  a  twenty-per-cent  solution.  In  Koch's  experiments  a  five-per-ceiit 
solution  failed  to  kill  anthrax  spores  in  ten  days.  Kills  pus  micro- 
cocci  in  two  hours  in  the  proportion  of  1  :  200  (Sternberg).  In  Bol- 
ton's  experiments  made  for  the  Committee  on  Disinfectants  of  the 
American  Public  Health  Association  the  following  results  were  ob- 
tained: Recent  cultures  in  bouillon,  time  of  exposure  two  hours  :  Ba- 
cillus of  typhoid  fever,  1  :  200;  cholera  spirillum,  1  :  500;  Bacillus  pyo- 
cyanus,  1 :200;  Brieger's  bacillus,  1 :200;  Emmerich's  bacillus,  1  :  200; 
Staphylococcus  pyogenes  aureus,  1  : 100  ;  Staphylococcus  pyogenes 
citreus,  1  : 100;  Staphylococcus  pyogenes  albus,  1  :  200;  Streptococcus 
pyogenes,  1  : 500.  When  ten  per  cent  of  dried  egg  albumin  was 
added  to  a  recent  culture  in  bouillon  of  the  typhoid  bacillus  the 
amount  required  to  insure  sterilization  was  1  : 10. 

In  the  report  of  the  Committee  on  Disinfectants  of  the  American 
Public  Health  Association  this  agent  is  recommended  in  "a  solu- 
tion of  two  to  five  per  cent  for  the  destruction  of  infectious  material 
not  containing  spores."  The  experimental  data  above  given  show 
that  this  is  a  liberal  allowance  for  material  which  does  not  contain 
an  excessive  amount  of  albumin.  In  the  experiments  of  Leitz  the 
typhoid  bacillus  in  cultures  was  destroyed  in  ten  minutes  by  a  five- 
per-cent  solution. 

Ferric  Chloride. — A  five-per-cent  solution  failed  in  two  days  to 
destroy  anthrax  spores,  but  was  effective  in  five  days  (Koch). 

Ferrous  Sulphate. — In  the  writer's  experiments  (1883)  a  solution 
of  twenty  per  cent  failed  to  destroy  micrococci  and  putrefactive  bac- 
teria. In  a  more  recent  experiment  ten  per  cent  failed  to  kill  pus 


186  ACTION  OP  SALTS. 


i,  but  was  fatal  to  Micrococcus  tetragenus—  two  hours'  exposure. 
K  .  >ch  found  that  a  five-per-cent  solution  failed  to  destroy  anthrax 
spores  in  six  days.  Exposure  to  a  twenty-per-cent  solution  for  forty- 
eight  hours  does  not  destroy  the  virus  of  symptomatic  anthrax  (Ar- 
loing,  Cornevin,  and  Thomas).  In  the  experiments  of  Jager  immer- 
sion in  a  solution  of  1  :  3  destroyed  the  infective  virulence  of  certain 
pathogenic  bacteria  (fowl  cholera,  rothlauf,  glanders),  as  tested  by 
injection  into  mice,  but  failed  to  kill  anthrax  spores  and  tubercle  ba- 
rilli.  The  antiseptic  power  of  ferrous  sulphate  is  placed  by  Miquel 
at  1  :  90.  In  the  writer's  experiments  1  :  200  prevented  the  develop- 
ment of  micrococci  and  of  putrefactive  bacteria  in  bouillon  placed 
in  the  incubating  oven  for  forty-eight  hours.  Leitz  found  that  a 
five-per-cent  solution  required  three  days'  exposure  for  the  destruc- 
tion of  the  typhoid  bacillus. 

Gold  Chloride.  —  Antiseptic  in  the  proportion  of  1  :  4,000  (Miquel). 
Boer  has  made  extended  experiments  with  the  chloride  of  gold  and 
sodium.  We  give  his  results  below.  In  his  disinfection  experi- 
ments a  bouillon  culture  which  had  been  in  the  incubating  oven  for 
t  \\  ••nty-four  hours  was  used,  and  the  time  of  exposure  was  two  hours. 


« 

Restrains 
development. 

Destroys 
vitality. 

Anthrax  bacillus  

1  :  40000 

1  :8000 

Diphtheria  bacillus  

1:40000 

1  :1000 

(Jlanders  bacillus  .          .... 

1  :  15000 

1  -400 

Typhoid  bacillus  

1  :  20000 

1  :500 

Cholera  spirillum        

1  •  25000 

1  :1000 

Lead  Chloride. — Antiseptic  in  the  proportion  of  1  :500  (Miquel). 

Lead  Nitrate. — Antiseptic  in  the  proportion  of  1  :  277  (Miquel). 

Lithium  (1l»l<tru1<>. — Antiseptic  in  the  proportion  of  1  : 11  (Mi- 
quel). 

Mangam  *<  rrotochloride. — Antiseptic  in  the  proportion  of  1:40 
(Miquel). 

Mercuric  Chloride.— Koch's  experiments  (1881)  gave  the  follow- 
ing results  :  A  solution  of  1 : 1,000  destroys  anthrax  spores  in  a  few 
minutes,  and  1  : 10,000  is  effective  after  a  more  prolonged  exposure. 
The  writer  (1884)  obtained  similar  results— 1  : 10,000  destroyed  the 
Bpo  res  of  Bacillus  anthracis  and  of  Bacillus  subtilis  in  two  hours. 
More  recent  experiments  indicate  that  failure  to  grow  in  culture  so- 
1  nt  ions  cannot  be  accepted  as  evidence  of  the  destruction  of  vitality 
in  the  case  of  spores  exposed  to  the  action  of  this  agent,  unless  due 
pi  "cautions  ;m>  t;ik«-n  to  exclude  the  restraining  influence  of  the  small 
amount  of  mercuric  chloride  which  remains  attached  to  the  spores. 
l,:,,i  ;l-<  .-itainrd  that  the  development  of  spores  is  restrained  by 


ACTION   OF   SALTS.  187 

the  presence  of  1  : 300,000  in  a  culture  medium,  and  Geppert  has  re- 
cently shown  that  even  so  small  an  amount  as  1  :  2,000,000  will  pre- 
vent the  development  of  spores  the  vitality  of  which  has  been  reduced 
by  the  action  of  a  strong  solution  (1  : 1,000).  When  this  restraining 
action  is  entirely  neutralized  by  washing  the  spores  in  a  solution  con- 
taining ammonium  sulphide  it  requires,  according  to  Geppert,  a  solu- 
tion of  1:1,000  acting  for  one  hour  to  completely  destroy  the  vitality 
of  anthrax  spores.  Frankel  found  that  a  solution  of  1  : 1,000  was 
effective  in  half  an  hour.  The  typhoid  bacillus,  the  bacillus  of  mouse 
septicaemia,  and  the  cholera  spirillum,  in  bouillon  cultures  and  in 
cultures  in  flesh-peptone-gelatiii,  are  destroyed  in  two  hours  by 
1  : 10,000  ;  but  in  a  bouillon  culture  to  which  ten  per  cent  of  dried 
egg  albumin  was  added  a  one-per-cent  solution  was  required  to  de- 
stroy the  typhoid  bacillus  in  the  same  time  (Bolton).  According  to 
Van  Ermengem,  cultures  of  the  cholera  spirillum  in  bouillon  are  steril- 
ized in  half  an  hour  by  1  :  60,000,  but  cultures  in  blood  serum  require 
1  :  800  to  1  : 1,000.  In  experiments  upon  tuberculous  sputum  Schill 
and  Fischer  found  that  exposure  of  fresh  sputum  to  an  equal  amount 
of  a  1  : 2,000  solution  for  twenty -four  hours  failed  to  disinfect  it,  as 
shown  by  inoculation  experiments  in  guinea-pigs.  The  antiseptic 
power  of  mercuric  chloride  is  given  by  Miquel  as  1  : 14,300.  In  the 
writer's  experiments  1  : 33,000  was  found  to  prevent  the  development 
of  putrefactive  bacteria  in  bouillon,  but  a  minute  bacillus  contained  in 
broken-do wii  beef  infusion  multiplied,  after  several  days,  in  1  : 20,000. 
The  pus  cocci  were  restrained  in  their  development  by  1  :  30,000. 

In  Behring's  experiments  the  anthrax  bacillus  and  cholera  spiril- 
lum were  killed  in  one  hour  by  1  : 100,000  when  the  temperature 
was  36°  C.,  but  at  a  temperature  of  3°  C.  the  proportion  required 
was  1 : 25,000.  The  same  author  states  that  at  22°  C.  Staphylo- 
coccus  aureus  in  bouillon  is  not  always  killed  in  twenty-five  minutes 
by  1 : 1,000. 

In  a  recent  series  (1891)  of  experiments  Abbott  has  shown  that  a 
1  : 1,000  solution  does  not  always  destroy  Staphylococcus  pyogenes 
aureus  in  five  minutes.  He  says:  "Frequently  all  the  organisms 
would  be  destroyed  after  five  minutes'  exposure,  but  almost  as  often 
a  certain  few  would  resist  for  that  length  of  time,  and  even  longer, 
going  in  some  cases  to  ten,  twenty,  and  even  thirty  minutes. " 

According  to  Yersin,  a  solution  of  1  : 1,000  kills  the  tubercle  bacil- 
lus in  one  minute. 

We  might  add  considerably  to  the  experimental  data  given,  but 
the  results  already  recorded  are  sufficient  to  show  the  value  of  this 
agent  as  an  antiseptic  and  germicide,  and  justify  its  use  for  general 
purposes  of  disinfection  in  the  proportion  of  1  : 500  or  1  :  1,000  for 
material  containing  spores,  and  in  the  proportion  of  1  : 2,000  to 


Ihtf 


ACTION   OF   SALTS. 


1  :  :>,000  for  pathogenic  bacteria  in  the  absence  of  spores;  due  regard 
}>eing  had  to  the  fact  that  the  presence  of  albumin  very  materially 
reduces  its  germicidal  potency,  and  that  it  may  be  decomposed  and 
neutralized  by  alkalies  and  their  carbonates,  by  hydrosulphuric  acid, 
and  by  many  other  substances. 

The  albuminate  of  mercury,  as  has  been  shown  by  Lister,  is  solu- 
ble in  an  excess  of  albumin,  and,  according  to  Behring,  is  just  as 
effective  as  an  aqueous  solution  containing  the  same  amount  of  sub- 
limate when  dissolved  in  an  albuminous  liquid  like  blood  serum  (?). 

In  practice  the  addition  of  a  mineral  acid  to  sublimate  solutions, 
or  of  sodium,  potassium,  or  ammonium  chloride,  is  to  be  recom- 
mended, to  prevent  the  precipitation  of  the  mercuric  chloride  by  al- 
bumin in  fluids  containing  it.  Behring  recommends  the  addition 
of  five  parts  of  sodium  or  potassium  chloride  to  one  of  the  subli- 
mate. Such  a  solution  is  more  stable  than  a  simple  solution  of  sub- 
limate, and  no  precipitate  is  formed  by  the  addition  of  alkalies  or  by 
albumin. 

The  same  result  is  obtained,  according  to  La  Place,  by  the  addi- 
tion of  five  parts  of  hydrochloric  or  tartaric  acid  to  one  part  of  sub- 
limate in  aqueous  solution. 

Mercuric  Cyanide,  Hg(CN)2,  and  the  Oxycyanide  of  mercury 
have  been  tested,  with  the  following  results  :  Staphylococcus  aureus 
is  destroyed  in  five  minutes  by  1  : 100,  in  one  hour  by  1  : 1,000,  in 
two  hours  by  1  : 1,500  (Chibret).  The  development  of  Bacillus  an- 
thracis  in  culture  solutions  is  prevented  by  the  presence  of  cyanide 
of  mercury  in  the  proportion  of  1  :  25,000,  and  by  the  oxy cyanide  by 
1  : 16,000  (Behring). 

Boer  obtained  the  following  results  with  the  oxycyanide — cul- 
turos  in  bouillon,  twenty-four  hours  in  incubating  oven,  time  of 
exposure  two  hours  : 


Restrained 
development. 

Destroyed 
vitality. 

Anthrax  bacillus  

•  80000 

1  •  40000 

Diphtheria  bacillus  

•  soooo 

i  .  4.0000 

Glanders  bacillus  

•  fiOOOO 

1  •  '^0000 

Typhoid  bacillus  

•  60000 

1  •  SOOOO 

Cholera  spirillum  

•  'Mini  KI 

1  •  ftoooo 

M<  rcuric  Iodide.— The  antiseptic  value  of  this  salt  is  placed  by 
Miquel  at  1  : 40,000,  which  is  more  than  double  that  given  by  the 
^;mie  author  to  the  Im-hloride.  In  the  writer's  experiments  upon  the 
antiseptir  value  of  salts  and  oxides  of  mercury  the  following  results 
were  obtained  : 


ACTION   OF   SALTS.  189 


Active. 

Failed. 

Biniodide  of  mercury  

1  •  20000 

1  •  40000 

Bichloride  

1    15000 

1  •  20000 

Protiodide  

1    10000 

1  •  20000 

Yellow  oxide  

1  :  1000 

1    2000 

Black  oxide  

1  -500 

1  •  1000 

Morphia  Htjdrochlorate. — Antiseptic  in.  the  proportion  of  1  : 13 
(Miquel). 

Nickel  Sulphate. — Antiseptic  in  the  proportion  of  1  : 400  (Mi- 
quel). 

Platinum  Bichloride. — Antiseptic  in  the  proportion  of  1  :  3,333 
(Miquel). 

Potassium  Acetate. — A  saturated  solution  of  this  salt  failed  to 
kill  anthrax  spores  in  ten  days  (Koch). 

Potassium  Arsenite. — In  the  writer's  experiments  Fowler's  solu- 
tion failed  to  kill  micrococci  in  two  hours  in  the  proportion  of  four 
per  cent.  Miquel  places  the  antiseptic  value  of  potassium  arsenite 
at  1  :  8. 

Potassium  Bichromate. — A  five-per-cent  solution  failed  in  two 
days  to  destroy  anthrax  spores  (Koch).  Efficient  as  an  antiseptic  in 
the  proportion  of  1  :  909  (Miquel). 

Potassium  Bromide. — The  bacillus  of  typhoid  fever  and  the 
cholera  spirillum  fail  to  grow  in  culture  solutions  containing  9  to 
10.6  per  cent,  and  are  killed  in  four  or  five  hours  by  ten  to  twelve 
per  cent  (Kitasato). 

Potassium  Carbonate. — The  development  of  the  typhoid  bacil- 
lus and  of  the  cholera  spirillum  is  prevented  by  0.74  to  0.81  per 
cent,  and  these  bacteria  are  killed  in  five  hours  by  1  per  cent  (Kita- 
sato). 

Potassium  Chlorate. — In  the  writer's  experiments  a  four-per- 
cent solution  failed  in  two  hours  to  kill  Micrococcus  Pasteuri.  A 
five-per-cent  solution  failed  in  six  days  to  destroy  anthrax  spores 
(Koch). 

Potassium  Chromate. — A  five-per-cent  solution  failed  to  kill 
anthrax  spores  in  five  days  (Koch). 

Potassium  Cyanide. — Antiseptic  in  the  proportion  of  1  :  909 
(Miquel). 

Potassium  Iodide. — A  solution  of  five  per  cent  does  not  destroy 
anthrax  spores  in  eighty  days  (Koch).  Putrefactive  bacteria  in 
broken-down  beef  infusion  are  not  destroyed  by  two  hours'  exposure 
in  a  twenty-per-cent  solution  (Sternberg).  The  typhoid  bacillus  and 
the  cholera  spirillum  do  not  grow  in  culture  solutions  containing 


190 


ACTION   OF   SALTS. 


eight  per  cent,  and  are  destroyed  by  five  hours'  exposure  to  9.23  per 
cent'(Kitasato).  Antiseptic  in  the  proportion  of  1  :  7  (Miquel). 

Potaasium  Permanganate.— In  the  writers  experiments  (1881) 
a  two-per-cent  solution  was  required  to  destroy  Micrococcus  Pasteuri 
in  the  blood  of  a  rabbit.  In  later  experiments  pus  cocci  in  bouillon 
were  killed  by  1  : 833— time  of  exposure  two  hours.  One  per  cent 
was  found  by  Koch  not  to  destroy  anthrax  spores  in  two  days,  but 
five  per  cent  was  effective  in  one  day.  The  glanders  bacillus  is  de- 
stroyed in  two  minutes  by  a  one-per-cent  solution  (Loflfler).  The 
experiments  of  Jager  show  that  a  one-per-cent  solution  is  not  reli- 
able for  the  destruction  of  anthrax  bacilli  and  other  pathogenic  bac- 
teria tested,  but  a  five-per-cent  solution  was  effective.  The  tubercle 
bacillus  was  not,  however,  killed  by  exposure  in  a  five-per-cent  solu- 
tion. According  to  Miquel,  permanganate  of  potash  is  an  antiseptic 
in  the  proportion  of  1  :  285. 

Quinine  Hydrobromate. — Antiseptic  in  the  proportion  of  1  : 182 
(Miquel). 

Quinine  Hydrochlorate. — Antiseptic  in  the  proportion  of  1  :  900 
(Ceri).  Quinine  dissolved  with  hydrochloric  acid  destroys  anthrax 
spores  in  ten  days  in  one-per-cent  solution  (Koch). 

Quinine  Sulphate. — The  writer  found  that  in  the  proportion  of 
1  :  800  quinine  prevents  the  development  of  various  micrococci  and 
l»;tcilli.  A  ten-per-cent  solution  does  not  destroy  the  bacilli  of  symp- 
tomatic anthrax  (Arloing,  Cornevin,  and  Thomas). 

Silver  Nitrate. — Miquel  places  nitrate  of  silver  next  to  mercuric 
chloride  as  an  antiseptic,  effective  in  the  proportion  of  1  : 12,500. 
Behring  also  places  it  next  to  bichloride  as  an  antiseptic  and  germi- 
cide, and  says  that  it  is  even  superior  to  this  salt  in  albuminous 
fluids.  He  reports  that  it  prevents  the  development  of  anthrax 
spores  when  present  in  a  culture  liquid  in  the  proportion  of  1 : 80,000, 
and  in  the  proportion  of  1  : 10,000  destroys  these  spores  in  forty- 
eight  hours.  We  give  below  the  result  of  recent  experiments  by 
Boer,  in  which  the  time  of  exposure  was  two  hours  : 


Restrains 
development. 

Destroys 
vitality. 

•  600^0 

.  20000 

Dipliflicriu  liarilius  

•  60000 

•  2500 

•  75000 

•  4000 

Typhoid  bacillus  

•  50000 

•4000 

(  'Imlrrii  spirillum  

•  50000 

•  4000 

Silver  Chloride.— A.  solution  of  chloride  of  silver  in  hyposulphite 
of  soda  is  much  less  effective  as  an  antiseptic  than  nitrate  of  silver. 


ACTION   OF    SALTS.  191 

Behring  found  that  to  prevent  the  development  of  anthrax  spores  a 
solution  of  1  :  8,000  was  required. 

Sodium  Borate. — In  the  writer's  experiments  a  saturated  solu- 
tion of  borax  was  found  to  be  without  germicidal  power.  A  twenty- 
per-cent  solution  does  not  destroy  the  virus  of  symptomatic  anthrax 
(Arloing,  Cornevin,  and  Thomas).  A  five-per-cent  solution  failed 
to  destroy  anthrax  spores  in  fifteen  days  (Koch).  Antiseptic  in  the 
proportion  of  1  : 14  (Miquel). 

Sodium  Carbonate. — A  solution  of  2.2  per  cent  restrains  the 
growth  of  the  typhoid  bacillus,  and  of  2.47  per  cent  of  the  cholera 
spirillum.  The  first-named  bacillus  is  killed  by  four  or  five  hours' 
exposure  in  a  2. 47-per-cent  solution,  and  the  cholera  spirillum  by 
3.45  per  cent  (Kitasato). 

Sodium  Chloride. — A  saturated  solution  failed  in  forty-eight 
hours  to  destroy  the  virus  of  symptomatic  anthrax  (Arloing,  Corne- 
vin, and  Thomas).  A  saturated  solution  failed  in  forty  days  to  de- 
stroy anthrax  spores  (Koch).  A  saturated  solution  failed  in  twenty 
hours  to  destroy  the  tubercle  bacillus  in  fresh  sputum  (Schill  and 
Fischer).  In  the  writer's  experiments  a  five-per-cent  solution  failed 
to  kill  Micrococcus  Pasteuri  in  blood.  Antiseptic  in  the  proportion 
of  1  : 6  (Miquel).  According  to  Forster,  the  bacillus  of  typhoid 
fever,  the  bacillus  of  rouget,  and  the  streptococcus  of  pus  are  not 
killed  by  several  weeks'  exposure  in  strong  solutions  of  sodium  chlo- 
ride, but  the  cholera  spirillum  is  destroyed  in  a  few  hours.  Cultures 
of  the  tubercle  bacillus  are  not  sterilized  in  two  months  by  a  satu- 
rated solution  ;  and  tuberculous  organs  from  an  ox,  preserved  in  a 
solution  of  salt,  did  not  lose  their  power  of  infecting  susceptible  ani- 
mals inoculated  with  material  from  the  diseased  tissue.  The  flesh 
of  swine  which  died  of  rothlauf  was  found  by  Petri  to  still  contain 
the  bacillus  in  a  living  condition  after  having  been  preserved  in 
brine  for  a  month. 

Sodium  Hyposulphite. — In  the  writer's  experiments  a  saturated 
solution  failed  in  two  hours  to  kill  micrococci  and  bacilli.  Exposure 
for  forty-eight  hours  to  a  fifty-per-cent  solution  does  not  destroy  the 
virus  of  symptomatic  anthrax  (Arloing,  Cornevin,  and  Thomas). 
Antiseptic  in  the  proportion  of  1  :  3  (Miquel). 

Sodium  Sulphite. — The  results  with  a  saturated  solution  of  this 
salt  were,  in  the  writer's  experiments,  entirely  negative. 

Tin  Chloride. — A  one-per-cent  solution  acting  for  two  hours  de- 
stroyed the  bacteria  in  putrefying  bouillon,  while  0.8  per  cent  failed 
(Abbott). 

Zinc  Chloride.— In  the  writer's  experiments  1:200  destroyed 
Micrococcus  Pasteuri  in  two  hours,  but  a  two-per-cent  solution  was  re- 
quired to  kill  pus  cocci  in  the  same  time  ;  spores  of  Bacillus  anthracis 


192  ACTION  OF   SALTS. 

were  not  destroyed  by  two  hours'  exposure  in  a  ten-per-cent  solution, 
but  a  solution  of  five  per  cent  killed  tbe  spores  of  Bacillus  subtilis  in 
the  same  time.  Koch  found  that  anthrax  spores  germinated  after 
being  immersed  in  a  five-per-cent  solution  for  thirty  days.  The  de- 
velopment of  Bacillus  prodigiosus  is  only  slightly  retarded  by  expo- 
sure for  sixteen  hours  in  a  one-per-cent  solution.  Antiseptic  in  the 
proportion  of  1  : 526  (Miquel). 

Zinc  Sulphate. — In  the  writer's  first  experiments  a  twenty -per- 
cent solution  failed  to  destroy  in  two  hours  micrococci  obtained  from 
the  pus  of  an  acute  abscess.  In  later  experiments  a  micrococcus  from 
the  same  source  resisted  two  hours'  exposure  to  a  ten-per-cent  solu- 
tion, but  Micrococcus  tetragenus  was  destroyed  by  this  amount. 
Broken-down  beef  infusion  mixed  with  an  equal  quantity  of  a  forty- 
per-cent  solution  was  not  sterilized  after  two  hours'  contact.  In 
Koch's  experiments  anthrax  spores  were  found  to  germinate  after 
having  been  immersed  for  ten  days  in  a  five-per-cent  solution. 


XI. 

ACTION  OF  COAL-TAR  PRODUCTS,   ESSENTIAL 
OILS,   ETC. 

IN  the  present  section  we  shall  consider  the  action  upon  bacteria 
of  a  variety  of  organic  products,  and  for  convenience  will  arrange 
them  alphabetically. 

Acetone. — Anthrax  spores  grow  freely  after  two  days'  exposure 
to  the  action  of  this  agent;  at  the  end  of  five  days  their  development 
is  feeble  (Koch). 

Alcohol. — In  the  writer's  experiments  ninety-five-per-cent  alco- 
hol did  not  destroy  the  bacteria  (spores)  in  broken-down  beef  tea  in 
forty -eight  hours.  Micrococcus  Pasteuri  was  destroyed  by  two  hours' 
exposure  in  a  twenty-four-per-cent  solution  ;  pus  cocci  required  a 
forty -per-cent  solution.  Koch  found  that  absolute  alcohol  had  no 
effect  upon  anthrax  spores  exposed  to  its  action  for  one  hundred  and 
ten  days.  Schill  and  Fischer  found  that  when  tuberculous  sputum 
was  mixed  with  an  equal  amount  of  absolute  alcohol  its  infecting 
power  was  not  destroyed  in  twenty-four  hours,  but  that  in  the  pro- 
portion of  five  parts  to  one  of  sputum  it  was  effective  in  destroying 
the  tubercle  bacillus,  as  proved  by  inoculation  experiments.  Yersin 
found  that  in  pure  cultures  the  tubercle  bacillus  is  killed  by  five 
minutes'  exposure  to  the  action  of  absolute  alcohol. 

Aseptol  (orthophenol,  sulpho-carbolic  acid,  etc.). — This  substance 
is  freely  soluble  in  water.  According  to  Hueppe  a  three  to  five-per- 
cent solution  destroys  bacteria  in  the  absence  of  spores,  and  a  ten- 
per-cent  solution  destroys  anthrax  spores  in  ten  minutes. 

Aniline  Dyes. — Recent  researches  have  shown  that  some  of  the 
aniline  colors  possess  very  decided  germicidal  power.  Stilling  found 
that  solutions  of  methyl  violet  containing  1  : 30,000  exercise  a  re- 
straining influence  upon  the  development  of  putrefactive  bacteria 
and  pus  cocci,  and  that  these  microorganisms  are  destroyed  by  solu- 
tions containing  1  :  2,000  to  1  : 1,000.  Methyl  violet  has  been  placed 
in  the  market  by  Merck  under  the  name  of  pyoktanin.  Janicke  re- 
ports the  following  results  with  pyoktanin  :  Staphylococcus  pyogenes 
aureus  was  restrained  in  its  development  by  solutions  containing 
1  :  2,000,000,  Bacillus  anthracis  by  1  : 1,000,000,  Staphylococcus  pyo- 
genes by  1  :  333,300,  Spirillum  cholerae  Asiaticae  by  1  :  G2,500,  Bacil- 
lus ty  phi  abdominalis  by  1  : 5,000.  In  blood  serum  stronger  solutions 
13 


194 


ACTION  OF  COAL-TAR  PRODUCTS, 


were  required  (1 : 500,000  for  Staphylococcus  pyogenes  aureus).  Sta- 
phylococcus  pyogenes  aureus,  Streptococcus  pyogenes,  and  Bacillus 
anthracis  were  killed  in  thirty  seconds  by  1  : 1,000,  the  typhoid  bacil- 
lus by  the  same  amount  in  thirty  minutes.  Boer  found  malachite 
green  to  be  still  more  effective  than  methyl  violet.  In  his  experi- 
ments upon  bouillon  cultures  twenty-four  hours  old,  with  two  hours' 
exposure  to  the  action  of  the  disinfectant,  he  obtained  the  following 
results  : 

MALACHITE   GREEN. 


Restrains 
development. 

Destroys 
vitality. 

1  :  120000 

:  40000 

1  :  40000 

:8000 

1  :5'»00 

:300 

1  •  5000 

:300 

1  :  100000 

•5000 

METHYL  VIOLET   (PYOKTANIN). 


Restrains 
development. 

Destroys 
vitality. 

1  :  70003 

1  •  5000 

Diphtheria  bacillus  

1  :  10000 

1  •  2000 

1  •  2500 

1  •  150 

Typhoid  bacillus  

1  :2500 

1  -150 

1  *  30000 

1  -1000 

Aniline  Oil. — According  to  Riedlin,  the  addition  of  1  :  5  of  ani- 
line water  prevents  the  development  of  all  bacteria  in  nutrient  gelatin. 

Aromatic  Products  of  Decomposition. — Klein  has  tested  the 
germicidal  power  of  phenylpropionic  and  phenylacetic  acids.  He 
finds  that  anthrax  spores  resist  both  of  these  acids,  in  the  proportion 
of  1  : 400,  for  two  days,  but  in  the  absence  of  spores  anthrax  bacilli 
are  quickly  killed  by  a  solution  of  this  strength.  Certain  non-patho- 
genic micrococci  were  not  killed  by  exposure  for  twenty-five  minutes 
to  1  : 200.  The  caseous  matter  of  pulmonary  tuberculosis  infected 
guinea-pigs  after  exposure  for  ninety-six  hours  to  1  :  200. 

Aseptol. — A  ten-per-cent  aqueous  solution  kills  anthrax  spores  in 
ten  minutes,  and  a  three-  to  five-per-cent  solution  is  a  reliable  disin- 
fectant in  the  absence  of  spores  (Hueppe). 

Benzene,  C.H,. — Exposure  in  benzol  for  twenty  days  failed  to 
destroy  the  vitality  of  anthrax  spores  (Koch). 

Camphor.—  Alcohol  saturated  with  camphor  has  no  effect  upon 
the  virus  of  symptomatic  anthrax  (Arloing,  Cornevin,  and  Thomas) 

The  experiments  of  Cadeac  and  Meunier  show  that    camphor  (oil 


ESSENTIAL  OILS,    ETC.  195 

of,  or  tincture?)  has  but  little  germicidal  power.  The  typhoid  ba- 
cillus and  cholera  spirillum  were  only  destroyed  after  eight  to  ten 
days'  exposure  to  the  action  of  camphor  ("essence")* 

Carbolic  Acid. — Tested  upon  anthrax  spores,  Koch  found  a  one- 
per-cent  solution  to  be  without  effect  after  fifteen  days'  exposure  ;  a 
two-per-cent  solution  retarded  development  but  did  not  completely 
destroy  vitality  in  seven  days  ;  a  three-per-cent  solution  was  effec- 
tive in  two  days.  In  the  absence  of  spores  Koch  found  that  a  one- 
per-cent  solution  quickly  destroys  the  vitality  of  anthrax  bacilli. 
He  recommends  a  five-per-cent  solution  for  the  destruction  of  the 
"comma  bacillus"  in  the  discharges  of  cholera  patients,  and  a  two- 
per-cent  solution  for  the  disinfection  of  surfaces  soiled  with  such  dis- 
charges. In  the  writer's  experiments  1  :  200  destroyed  Micrococcus 
Pasteuri  in  two  hours  ;  and  pus  cocci  were  destroyed  by  1 : 125,  while 
1  : 200  failed.  Davaine  showed  by  inoculation  experiments  that  an- 
thrax bacilli  in  fresh  blood  are  destroyed  by  being  exposed  to  the 
action  of  a  one-per-cent  solution  for  one  hour.  A  two-per-cent  solu- 
tion destroys  the  dried  virus  of  symptomatic  anthrax  in  forty-eight 
hours  (Arloing,  Cornevin,  and  Thomas).  Solutions  in  oil  or  in  alco- 
hol have  been  shown  by  Koch  to  be  less  effective  than  aqueous  solu- 
tions. Thus  a  five-per-cent  solution  in  oil  failed  to  destroy  anthrax 
spores  in  one  hundred  and  ten  days,  and  the  same  solution  failed  to 
kill  the  bacilli,  in  the  absence  of  spores,  in  less  than  six  days.  A 
five-per-cent  solution  in  alcohol  did  not  destroy  anthrax  spores  in 
seventy  days.  Schill  and  Fischer  found  that  a  three-per-cent  solu- 
tion destroyed  the  infecting  power  of  tuberculous  sputum,  as  shown 
by  inoculation  into  guinea-pigs,  in  twenty-four  hours,  while  solutions 
of  one  and  two  per  cent  failed.  Bolton's  experiments  gave  the  fol- 
lowing results,  the  test  organisms  being  in  fresh  bouillon  cultures 
and  the  time  of  exposure  two  hours  :  The  cholera  spirillum,  the 
bacillus  of  typhoid  fever,  the  bacillus  of  schweinerothlauf,  Brieger's 
bacillus,  the  bacillus  of  green  pus,  and  the  pus  cocci  (Staphylococcus 
pyogenes  aureus,  albus,  and  citreus,  and  Streptococcus  pyogenes) 
were  all  killed  by  a  solution  of  one  per  cent,  while  in  a  majority  of 
the  experiments  a  one-half -per-cent  (1  : 200)  solution  failed.  Cul- 
tures of  the  typhoid  bacillus  in  flesh-peptone-gelatin  gave  the  same 
result  (1  : 100  with  two  hours'  exposure),  and  the  addition  of  ten  per 
cent  of  dried  egg  albumin  to  bouillon  cultures  did  not  influence  the 
result. 

The  experiments  of  La  Place  show  that  the  addition  of  hydro- 
chloric acid  to  a  disinfecting  solution  containing  carbolic  acid  greatly 
increases  its  germicidal  power  for  spores.  Thus  it  is  stated  that 
"  two  per  cent  of  crude  carbolic  acid  with  one  per  cent  of  pure  hydro- 
chloric acid  destroyed  anthrax  spores  in  seven  days,  while  two  per 
cent  of  carbolic  acid  or  one  per  cent  of  hydrochloric  acid  alone  did 


196  ACTION   OF   COAL-TAR   PRODUCTS, 

not  destroy  these  spores  in  thirty  days.  A  f our-per-cent  solution  of 
crude  carbolic  acid  with  two  per  cent  of  hydrochloric  acid  destroyed 
spores  in  less  than  an  hour  ;  four  per  cent  of  carbolic  acid  alone  did 
not  destroy  them  in  twelve  days.  Van  Ermengem  reports  that  in 
his  experiments  the  cholera  spirillum  in  chicken  bouillon  was  killed 
in  less  than  half  an  hour  by  1  :  600,  and  that  in  blood  serum  1  :  400 
was  effective.  Nicati  and  Rietsch  fix  the  germicidal  power  for  the 
cholera  spirillum  as  1  : 200,  the  time  of  exposure  being  ten  minutes  ; 
Ramon  and  Cajal,  1  : 50.  Boer  gives  the  following  results,  the  time 
of  exposure  being  two  hours,  cultures  in  bouillon  twenty-four  hours 
old: 


Restrains 
development. 

Destroys 
vitality. 

1  :750 

1  :  300 

Diphtheria  bacillus  

1  :500 

1  :  300 

1  :500 

1  :300 

1  :400 

1:200 

1  :600 

1  :400 

Leitz  reports  the  following  results  :  The  dejections  of  patients 
suffering  from  typhoid  fever,  mixed  in  equal  quantity  with  the  disin- 
fecting solution,  were  sterilized  by  a  five-per-cent  solution  of  car- 
l>olic  acid  in  three  days.  Pure  cultures  of  the  typhoid  bacillus  were 
sterilized  in  fifteen  minutes  by  a  five-per-cent  solution. 

In  the  experiments  of  Nocht  upon  anthrax  spores  it  was  found 
that  while  at  the  room  temperature  these  spores  were  not  destroyed 
by  several  days' exposure  in  a  five-per-cent  solution,  they  were  de- 
stroyed in  three  hours  by  the  same  solution  at  a  temperature  of  37.5°. 

Carbolic  acid  prevents  putrefactive  changes  in  bouillon  when  pre- 
-'•nt  in  the  proportion  of  1  :  333  (Miquel).  The  tubercle  bacillus  is 
killed  in  thirty  seconds  by  a  five-per-cent  solution,  and  in  one  minute 
by  a  one-per-cent  solution  (Yersin). 

Coffee  Infusion. — Experiments  have  been  made  by  Heim  and  by 
I  .mleritz  on  the  antiseptic  power  of  an  infusion  of  coffee.  The  first- 
nam.-.l  author  found  that  anthrax  bacilli  no  longer  developed  after 
tlm>e  hours*  exposure  in  a  ten-per-cent  solution,  but  spores  were  not 
kill.Ml  ; it  the  end  of  a  week.  Streptococci  in  a  bouillon  culture  re- 
•  iu  i  rod  twenty-four  hours'  exposure,  and  the  staphylococci  of  pus  were 
11.  >t  destroyed  in  this  time.  Liideritz  found  that  a  three-per-cent  iii- 
fiisi.m  restrained  the  growtli  in  nutrient  gelatin  of  the  typhoid  ba- 
rillu>.  ami  a  five-per-cent  infusion  killed  the  bacillus  in  two  days  ; 
the  rlinlrra  spirillum  failed  to  grow  in  presence  of  one  per  cent,  and 
a  solution  of  this  strength  killed  it  in  seven  hours  ;  Staphylococcus 


ESSENTIAL  OILS,    ETC.  197 

pyogenes  aureus  was  prevented  from  developing  by  two  per  cent, 
and  was  killed  in  six  days  by  a  five-per-cent  solution  ;  Streptococcus 
pyogenes  was  prevented  from  growing  by  one  per  cent,  and  killed  by 
a  ten-per-cent  solution  in  one  day  ;  Proteus  vulgaris  did  not  grow  in 
presence  of  2. 5  per  cent,  and  was  killed  in  two  days  by  ten  per  cent. 
The  question  as  to  what  constituent  of  the  infusion  of  roasted  coffee 
was  the  active  germicidal  agent  was  not  determined,  but  the  authors 
referred  to  agree  that  it  was  not  caffeine. 

Creolin. — This  is  a  coal-tar  product  which  resembles  crude  carbolic 
acid  in  appearance,  but  smells  rather  like  tar  than  like  phenol.  It 
makes  a  milky  emulsion  with  water,  which  has  been  proved  by  nu- 
merous experiments  to  possess  very  decided  germicidal  power,  being 
superior  to  carbolic  acid.  The  first  careful  test  of  the  germicidal 
power  of  this  agent  was  made  by  Esmarch,  who  found  that  a  solu- 
tion of  1 : 200  killed  the  cholera  spirillum  in  a  minute,  the  typhoid 
bacillus  at  the  end  of  several  days.  Anthrax  spores  were  not  de- 
stroyed in  twenty  days  by  a  five-per-cent  solution,  but  this  solution 
killed  the  tubercle  bacillus  attached  to  silk  threads  which  were  im- 
mersed in  it  for  a  short  time,  and  also  disinfected  tuberculous  sputum. 
Behring  has  shown  that  in  albuminous  liquids  creolin  is  less  effective 
than  carbolic  acid.  In  blood  serum  1  : 175  was  required  to  restrain 
the  development  of  staphylococci,  and  1  : 100  to  destroy  the  same  in 
ten  minutes.  Van  Ermengem,  as  a  result  of  numerous  experiments, 
arrived  at  the  conclusion  that  creolin  is  a  cheap  and  useful  disinfect- 
ing agent,  in  a  five-per-cent  solution,  for  various  pathogenic  organ- 
isms. Kaupe  reports  that  in  his  experiments  a  ten-per-cent  solution 
killed  anthrax  spores  in  twenty-four  hours.  According  to  Boer,  a 
solution  of  1 : 5,000  destroys  anthrax  bacilli  in  bouillon  cultures  in 
two  hours,  1 :2,000  diphtheria  bacilli,  1  :  300  the  glanders  bacillus, 
1 : 250  the  typhoid  bacillus,  and  1 : 3,000  the  cholera  spirillum. 

Creosote. — This  agent  was  found  by  the  writer  to  be  fatal  to 
micrococci  in  the  proportion  of  1  :  200.  In  the  proportion  of  one  per 
cent  it  failed,  after  twenty  hours'  exposure,  to  destroy  tubercle  ba- 
cilli in  sputum  (Schill  and  Fischer).  A  saturated  aqueous  solution 
does  not  destroy  the  tubercle  bacillus  in  cultures  in  twelve  hours 
(Yersin).  Guttman,  in  extended  experiments  upon  various  patho- 
genic organisms,  found  that  development  was  prevented  by  1  :  3,000 
to  1  :  4,000.  A  solution  containing  1  : 300  killed  Bacillus  pyocyarius 
and  Bacillus  anthracis  in  one  minute,  Bacillus  prodigiosus  in  two 
minutes,  and  the  Finkler-Prior  spirillum  in  one  minute  in  the  pro- 
portion of  1  : 600. 

Cresol. — This  is  a  dark,  reddish-brown,  transparent  fluid,  some- 
what thinner  than  creolin,  and,  like  it,  having  an  odor  of  tar.  It 
forms  an  emulsion  with  water,  which  is  not  so  stable  as  that  formed 


198  ACTION  OF  COAL-TAR   PRODUCTS, 

by  creolin.  Of  the  three  cresols,  ortho-,  meta-,  and  paracresol,  the 
second  was  found  by  Frankel  to  be  most  active.  This  author  states 
that  the  addition  of  sulphuric  acid  adds  greatly  to  its  germicidal 
|M»\ver.  A  four-per-cent  solution,  containing  equal  parts  of  cresol 
and  H,SO4,  killed  anthrax  spores  in  less  than  twenty-four  hours.  In 
Behring^s  experiments  a  solution  containing  ten  per  cent  of  each  killed 
anthrax  spores  in  eighty  minutes,  and  five  per  cent  of  each  in  one 
hundred  minutes,  while  an  eighteen-per-cent  solution  of  sulphuric 
acid  alone  did  not  kill  them  in  twenty -four  hours.  In  the  experi- 
ments of  Jager  a  two-percent  solution  destroyed  the  tubercle  bacillus 
in  cultures  and  in  sputum.  As  a  result  of  his  experiments  Behring 
concludes  that  cresol  has  no  advantage  over  carbolic  acid  as  a  ger- 
micide for  the  destruction  of  spores.  Tested  upon  Staphylococcus 
an  runs,  Streptococcus  erysipelatos,  and  Bacillus  pyocyanus,  Frankel 
found  that  a  solution  of  0. 3  per  cent  destroyed  these  microorganisms 
in  five  minutes,  while  a  two-per-cent  solution  of  carbolic  acid  re- 
quired fifteen  minutes'  contact  to  accomplish  the  same  result. 

Trikresol  (Schering)  has  been  tested,  with  favorable  results,  by 
several  bacteriologists.  According  to  Hammerl  it  is  about  twice  as 
active  a  germicide  as  carbolic  acid. 

Diaphtherin  (oxychinaseptol)  has  considerable  antiseptic  power, 
n>  shown  by  the  experiments  of  Rohrer  and  others.  Two  to  four 
drops  of  a  one-per-cent  solution  was  found  to  prevent  the  develop- 
ment of  test  organisms  (Staphylococcus  pyogenes  aureus  and  Bacillus 
anthracis)  in  twelve  cubic  centimetres  of  bouillon.  Stable  (1893) 
also  finds  that  as  an  antiseptic  it  is  far  superior  to  carbolic  acid  or  lysol, 
and  that  it  has  the  advantage  of  being  non-toxic.  Tested  upon  an- 
thrax spores  it  was  found  to  be  comparatively  inactive  as  a  germicide. 
A  fifteen-per-cent  solution  destroyed  anthrax  spores  in  three  days. 

Disinfektol—This  is  a  coal-tar  product  similar  to  creolin  which 
li  is  been  recommended  in  Germany  for  disinfecting  purposes.  It  is 
mi  oily,  dark-brown  fluid  having  a  specific  gravity  of  1. 086.  It  forms 
nn  emulsion  with  water,  which  has  a  slightly  alkaline  reaction.  It 
1  ms  been  tested  upon  typhoid  stools  by  Uffelmann  and  by  Beselin. 
The  last-named  author  gives  the  following  summary  of  the  results 
obtained  :  An  emulsion  of  five  per  cent  of  disinfektol  equals  in  value, 
f'»r  the  disinfection  of  the  liquid  discharges  of  typhoid  patients,  12.5 
per  cent  of  creolin,  thirty-three  percent  of  hydrochloric  acid,  five  per 
cent  of  carbolic  acid,  1 : 500  of  mercuric  chloride. 

Ether. — Anthrax  spores  may  germinate  after  being  immersed  in 
-il|»huric  ether  for  eight  days  (Koch).  The  tubercle  bacillus  is  de- 

•yed  by  ten  minutes'  exposure  to  the  action  of  ether  (Yersin). 

/•:,s-.s-r nhtil  Oils.  —Chamberlain  has  made  an  extended  series  of 
experiments  to  determine  the  antiseptic  power  of  the  vapor  of  vola- 


ESSENTIAL  OILS,    ETC.  199 

tile  oils.  A  large  number  of  essential  oils  tested  were  found  to  pre- 
vent the  development  of  the  anthrax  bacillus,  while  a  few  did  not. 
At  the  end  of  six  days  the  tubes  were  opened  and  the  oil  absorbed  by 
the  culture  liquid  allowed  to  evaporate.  Cultures  were  now  obtained 
from  all  except  the  following,  which,  it  was  inferred,  had  destroyed 
the  vitality  of  the  spores  :  Angelica,  cinnamon  of  China,  cinnamon 
of  Ceylon,  geranium  of  France,  geranium  of  Algeria,  origanum. 

Cadeac  and  Meunier  have  also  made  extended  experiments  upon 
the  typhoid  bacillus  and  the  bacillus  of  glanders,  for  the  purpose  of 
determining  the  germicidal  power  of  agents  of  this  class.  Their 
method  consisted  in  the  introduction  of  a  sterilized  platinum  needle 
into  a  pure  culture  of  the  test  organism,  in  immersing  it  in  the 
essential  oil  for  a  certain  time,  and  then  making  with  it  a  puncture 
in  a  suitable  solid  culture  medium.  Their  results  are  given  below 
for  the  typhoid  bacillus. 

Essences  which  kill  the  bacillus  after  a  contact  of  less  than 
twenty-four  hours : 

At  the  end  of— 

Cinnamon  of  Ceylon,       .  .  .  .  .12  minutes. 

Cloves,  ......  25 

Eugenol,    .......      30 

Thyme,  ......  35 

Wild  thyme,          . 35 

Verbena  of  India,       .....  45 

Geranium  of  France,        .  .  .  .  .50 

Origanum,       ......  75 

Patchouly,  ......      80 

Zedoary,  ......  2  hours. 

Absinthe 4      u 

Sandal  wood,    .  .  .  .  .  .  12      " 

The  following  were  effective  in  from  twenty-four  to  forty-eight 
hours:  Cumin,  caraway,  juniper,  matico,  galbanum,  valerian,  citron, 
angelica,  celery,  savin,  copaiba,  pepper,  turpentine,  opoponax,  rose, 
chamomile  ;  the  following  required  from  two  to  four  days:  Illicium, 
sassafras,  tuberose,  coriander;  the  following  from  four  to  eight  days: 
Calamus,  sage,  fennel,  mace,  cascarilla,  orange  of  Portugal;  the  fol- 
lowing in  eight  to  ten  days  :  Mint,  nutmeg,  rosemary,  carrot,  mus- 
tard, anise,  onion,  marjoram,  bitter  almonds,  cherry  laurel,  myrtle, 
lavender,  eucalyptus,  cedar,  cajuput,  wintergreen,  camphor. 

Riedlin  reports  as  the  result  of  his  experiments  that  the  essential 
oils  which  have  the  greatest  antiseptic  value  are  oil  of  lavender,  eu- 
calyptus, rosemary,  and  cloves. 

Eucalyptol. — Chabaunes  and  Perret  found  that  a  five-per-cent 
solution  of  eucalyptol  is  without  effect  upon  tubercle  bacilli  in  spu- 
tum. According  to  Behring,  eucalyptol  is  about  four  times  less  ac- 
tive as  a  disinfectant  than  carbolic  acid.  * 


200  ACTION   OF  COAL-TAR   PRODUCTS, 

Euphorin  (Phenylurethan)  has  been  tested  by  Colasanti  (1894), 
who  finds  that  it  has  rather  feeble  germicidal  activity. 

Formaldehyde  (formol,  formalin)  has  very  decided  germicidal 
power.  According  to  Pottevin  (1894)  in  the  absence  of  spores  a  solu- 
tion of  1 : 1,000  kills  bacteria,  in  comparatively  small  numbers,  in  from 
fifteen  minutes  to  several  hours.  For  the  destruction  of  spores  a 
much  stronger  solution  is  required — a  fifteen-per-cent  solution  at 
15°  C.  killed  anthrax  spores  in  one  and  one-half  hours,  and  spores 
of  Bacillus  subtilis  in  twenty  hours.  At  higher  temperatures  the 
germicidal  action  is  more  energetic,  and  microorganisms  exposed  to 
the  vapor  of  formol  are  very  quickly  destroyed.  Vanderlinden  and 
de  Buck  (1895)  find  that  solutions  of  formalin  are  decidedly  inferior 
to  corresponding  solutions  of  carbolic  acid,  creolin,  or  solveol,  and  are, 
too  irritating  to  be  used  in  surgical  practice.  They  report  that  a 
solution  of  five  per  cent  failed  to  destroy  their  test  organisms — 
Bacillus  coli  communis,  Bacillus  typhi  abdominalis,  Staphylococcus 
pyogenes  aureus.  Experiments  made  by  Reed,  at  the  Army  Medical 
Museum  in  Washington,  show  that  the  diphtheria  bacillus  and  other 
test  organisms  are  quickly  killed  by  formalin  vapor. 

Glycerin  has  no  action  upon  the  virus  of  symptomatic  anthrax 
(Arloing,  Cornevin,  and  Thomas),  and  is  inert  as  regards  the  spores 
of  anthrax  (Koch).  Glycerin  prevents  putrefactive  decomposition  in 
bouillon  when  present  in  the  proportion  of  1 :4  (Miquel).  Roux  has 
shown  that  the  addition  of  five  per  cent  of  glycerin  to  a  culture 
medium  is  favorable  to  the  growth  of  the  tubercle  bacillus ;  it  is  also 
appropriated  as  pabulum  by  various  other  species. 

Ouaiacol. — Kuprianow,  as  a  result  of  extended  experiments  with 
this  agent  (1894),  reports  that  it  ranks  below  cresol  and  carbolic  acid 
flfefi  germicide.  In  the  proportion  of  1 : 500  it  restrains  the  develop- 
ment of  the  cholera  spirillum,  and  the  author  named  suggests  its  in- 
ternal administration  in  this  disease  on  account  of  its  non-toxic  and 
non-irritant  properties. 

Hydroxylamin.— Heinisch  found  that  the  development  of  the 
anthrax  bacillus  is  prevented  by  1 : 77  of  hydroxylamin  hydro- 
chlorate,  and  of  the  diphtheria  bacillus  by  1 :  75.  In  these  experiments 
a  solution  of  soda  was  added  to  release  the  hydroxylamin.  Marp- 
mann  found  that  1:100  preserved  milk  without  change  for  four 
to  six  weeks,  and  that  alkaline  fermentation  of  urine  was  prevented 
by  1:1,000. 

Ichthyol.—  Latteux  (1892)  reports  that  the  various  pathogenic 
bacteria  used  by  him  as  test  organisms  were  killed  by  a  five-per-cent 
solution  (time  ?)  with  exception  of  Streptococcus  pyogenes,  which 
required  a  six  to  seven-per-cent  solution.  The  more  recent  experi- 
ments of  Abel  (1893)  gave  less  favorable  result,  but  the  agent  was 


ESSENTIAL   OILS,    ETC.  201 

shown  to  have  considerable  antiseptic  value — 1 :  2,000  restrained  the 
development  of  streptococci;  1:  500  of  the  diphtheria  bacillus ;  1:  20 
of  Staph ylococcus  pyogenes  aureus ;  1 :  33  the  bacillus  of  typhoid 
fever.  Streptococci  and  diphtheria  bacilli  were  destroyed  in  twenty- 
four  hours  by  a  solution  of  1 :  200 ;  Staphylococcus  aureus,  subjected 
to  the  action  of  pure  ichthyol,  was  destroyed  in  five  hours — in  a  five- 
per-cent  solution  it  survived  for  four  days.  Cultures  of  the  typhoid 
bacillus  mixed  with  a  fifty-per-cent  solution  were  not  completely 
sterilized  in  thirty  hours;  a  small  number  of  bacilli  in  bouillon  were, 
however,  destroyed  by  a  three-per-cent  solution  in  forty-eight  hours. 
Anthrax  spores  on  silk  threads  were  not  destroyed  by  a  fifty-per-cent 
solution  at  the  end  of  one  hundred  and  forty  days. 

Indol. — When  added  in  excess  to  water  this  agent  failed  to  de- 
stroy anthrax  spores  in  eighty  days  (Koch). 

Izal  is  a  coal-tar  product  which  has  recently  been  introduced  as 
a  disinfectant.  Klein  (1892)  reports  that  in  the  strength  of  ten  per 
cent  it  kills  anthrax  spores  in  fifteen  minutes.  In  the  absence  of 
spores  various  pathogenic  bacteria  were  killed  in  five  minutes  by  a 
solution  containing  1 :  200. 

Lanolin. — According  to  Gottstein,  various  microorganisms  tested 
by  him  failed  to  grow  in  cultures  after  having  been  in  contact  with 
pure  lanolin  for  five  to  seven  days. 

Loretin. — Korff  (1895)  claims  for  this  agent  that  a  two-per-cent 
solution  is  superior  to  corresponding  solutions  of  lysol,  metakresol, 
or  phenol,  and  that  it  has  the  advantage  of  being  non-toxic,  odorless, 
and  non-irritating. 

Lysol. — Weiss  (1895)  has  tested  this  product  and  reports  that  a 
solution  of  three-fourths  per  cent  destroyed  his  test  organisms  (pus 
cocci,  typhoid  bacillus,  Bacillus  coli  communis,  etc.)  in  five  minutes. 
Anthrax  spores  were  destroyed  by  the  same  solution  in  one  hour. 

Naphthol. — In  the  proportion  of  1 :  10,000  naphthol  prevents  the 
development  of  the  glanders  bacillus,  the  anthrax  bacillus,  the  typhoid 
bacillus,  the  micrococcus  of  fowl  cholera,  of  Staphylococcus  aureus 
and  albus,  and  of  several  other  microorganisms  tested  by  Maximo- 
vitch.  The  same  author  states  that  although  insoluble  in  cold  water, 
water  at  70°  C.  dissolves  0.44  in  one  thousand  parts.  When  urine  is 
shaken  up  with  naphthol  in  powder  it  does  not  undergo  fermenta- 
tion. 

In  the  experiments  of  Foote  hydronaphthol  was  found  to  show 
some  germicidal  power  in  the  proportion  of  1 :  2,300,  but  the  conclu- 
sion is  reached  that  a  saturated  aqueous  solution  (1 :  1,150)  does  not 
equal  a  one-per-cent  solution  of  carbolic  acid  or  of  creolin. 

The  writer,  in  1892,  obtained  the  following  results  in  experiments 
with  naphthols  upon  the  cholera  spirillum. 


202  ACTION  OF  COAL-TAR  PRODUCTS, 

Alpha-naphthol  and  beta-naphthol  have  about  the  same  antiseptic  and 
germicidal  value.  In  the  proportion  of  1  : 16,000  both  prevent  the  develop- 
ment of  the  cholera  spirillum  in  peptonized  beef -tea,  while  1  :  24,000  fails  to 
pi-event  development.  In  the  proportion  of  1  :  3,000  both  destroy  the  vital- 
ity of  the  cholera  spirillum  in  bouillon  cultures,  twenty-four  hours  old, 
after  two  hours'  contact,  while  1 :  4,000  fails  to  destroy  this  microorganism 
in  the  time  mentioned — two  hours. 

In  experiments  made  with  a  solution  of  1  : 1,000,  added  to  an  equal 
quantity  of  a  twenty-four  hours  old  bouillon  culture — making  1  :  2,000  after 
mixture — and  in  which  the  time  of  contact  varied  from  live  to  thirty  minutes, 
alpha-,  beta-,  and  hydronaphthol  were  found  to  destroy  the  cholera  germ  by 
fifteen  minutes'  exposure,  but  to  fail  after  ten  minutes'  contact,  so  that  the 
germicidal  value  of  each  of  these  is  similar,  or  nearly  so. 

In  all  these  experiments  the  line  was  sharply  drawn  between  success  and 
failure.  No  development  occurred  and  the  bouillon  remained  transparent 
in  those  experiments  in  which  the  germicidal  action  was  complete,  and  a 
characteristic  development  occurred  within  twenty-four  hours  in  those  ex- 
periments in  which  there  was  a  failure  to  destroy  the  spirillum. 

Benzo-naphthol  has  110  germicidal  power,  probably  because  it  is  insoluble 
in  water.  At  least  this  is  my  inference  from  the  experiments  made.  One 
gamme  was  added  to  one  thousand  cubic  centimetres  of  distilled  water,  and 
after  vigorous  shaking  was  placed  in  the  steam  sterilizer  for  half  an  hour. 
At  the  end  of  this  time  the  greater  portion,  at  least,  of  the  beiizo-naphthol  re- 
mained undissolved  at  the  bottom  of  the  flask.  The  saturated  solution  (?) 
was  then  filtered  and  added  to  recent  bouillon  cultures  of  the  cholera  spiril- 
lum in  the  proportion  of  1  : 1,  1 :  2,  1 :  4,  and  2:1.  At  the  end  of  two  hours 
sterile  bouillon  in  test  tubes  was  inoculated  from  each  of  these  and  placed  in 
the  incubating  oven.  At  the  end  of  forty-eight  hours  a  characteristic  devel- 
opment of  the  cholera  spirillum  had  occurred  in  all  of  the  tubes. 

Olive  Oil. — Anthrax  spores  germinate  after  having  been  im- 
mersed for  ninety  days  in  pure  olive  oil  (Koch). 

Oil  of  Mustard. — Koch  found  that  the  development  of  anthrax 
spores  is  prevented  by  1 : 33,000. 

Oil  of  Peppermint.— A.  five-per-cent  solution  in  alcohol  failed  in 
twelve  days  to  destroy  anthrax  spores,  but  the  development  of  these 
spores  is  restrained  by  1 :  33,000  (Koch). 

Oil  of  Turpentine  destroys  anthrax  spores  in  five  days,  but  failed 
to  do  so  in  one  day  (Koch) .  The  development  of  anthrax  spores  is 
prevented  by  1 :  75,000  (Koch).  The  addition  of  1 :  200  to  nutrient 
gelatin  prevents  the  development  of  bacteria  (Riedlin) .  An  excess 
of  oil  of  turpentine  added  to  a  liquefied  gelatin  culture  of  Staphylo- 
ooccus  aureus  does  not  destroy  this  micrococcus  in  five  hours  (v. 

<  liiistmas-Dirckinck-Holmfeld). 

Saprol. — Laser  (1802)  recommends  this  agent  for  the  disinfection 

<  >t  t  he  excreta  of  cholera  and  typhoid  patients.     He  reports  that  in  the 
proportion  of  1  :  100  it  sterilizes  liquid  faeces  in  twenty-four  hours. 

Skatol  in  excess  in  water  has  no  germicidal  power,  as  tested  upon 
anthrax  spores  (Koch). 

smofo?. — The  researches  of  Beu  show  that  meats  which  have  been 
preserved  by  smoking  commonly  contain  living  bacteria  capable  of 
growing  in  culture  media;  and  Petri  has  shown  that  pork  which  has 


ESSENTIAL   OILS,    ETC.  203 

been  salted  for  a  month  and  then  smoked  for  fourteen  days  may  still 
contain  the  bacillus  of  rothlauf  in  a  living  condition,  as  shown  by  in- 
oculation experiments.  It  was  not  until  about  six  months  after  smok- 
ing that  the  bacillus  failed  to  give  evidence  of  vitality. 

Thymol. — A  five-per-cent  solution  in  alcohol  does  not  destroy 
anthrax  spores  in  fifteen  days,  but  the  development  of  these  spores 
is  retarded  by  a  solution  of  1  : 80,000  (Koch).  The  anthrax  bacillus 
and  staphylococci  fail  to  grow  in  culture  media  containing  1  :  3,000 
(Samter).  The  tubercle  bacillus  is  destroyed  by  contact  with  thy- 
mol for  three  hours  (Yersin).  Thymol  has  about  four  times  less 
germicidal  power  than  carbolic  acid  (Behring).  Antiseptic  in  the 
proportion  of  1  : 1,340  (Miquel). 

Tobacco  Smoke. — Tassinari  found  that  tobacco  smoke  restrains 
the  development  of  bacteria,  and  that  certain  species  failed  to  de- 
velop after  exposure  for  half  an  hour  in  an  atmosphere  of  tobacco 
smoke — spirillum  of  cholera  and  Friedlander's  bacillus. 


XII. 

ACTION  OF  BLOOD  SERUM  AND   OTHER  ORGANIC 

LIQUIDS. 

Blood  Serum.— Bacteriologists  have  long  been  aware  of  the  fact 
that  many  species  of  bacteria,  when  injected  into  the  circulation  of  a 
living  animal,  soon  disappear  from  the  blood,  and  that  the  blood  of 
such  an  animal  a  few  hours  after  an  injection  of  putrefactive  bacte- 
ria, for  example,  does  not  contain  living  bacteria  capable  of  develop- 
ing in  a  suitable  nutrient  medium.  Wyssokowitsch,  in  an  extended 
series  of  experiments,  has  shown  that  non-pathogenic  bacteria  in- 
jected into  the  circulation  may  be  obtained  in  cultures  from  the  liver, 
spleen,  kidneys,  and  bone  marrow  after  they  have  disappeared  from 
the  blood,  but  that,  as  a  rule,  those  present  in  these  organs  have  lost 
their  vitality,  as  shown  by  culture  experiments,  in  a  period  varying 
from  a  few  hours  to  two  or  three  days.  According  to  the  theory  of 
Metschnikoff,  this  destruction  of  bacteria  in  the  blood  and  tissues  of  a 
living  animal  is  effected  by  -the  cellular  elements,  and  especially  by 
the  leucocytes,  which  pick  up  and  digest  these  vegetable  cells  very 
much  as  an  amoeba  disposes  of  similar  microorganisms  which  serve 
it  as  food.  Some  such  theory  seemed  necessary  to  account  for  the 
disappearance  of  bacteria  from  the  blood  before  the  demonstration 
was  made  that  the  serum  of  the  circulating  fluid,  quite  indepen- 
dently of  its  cellular  elements,  possesses  very  decided  germicidal 
power. 

Von  Fodor  first  (1887)  called  attention  to  the  fact  that  anthrax  ba- 
Hlli  maybe  destroyed  by  freshly  drawn  blood ;  and  Nuttall  (1888), 
in  an  extended  series  of  experiments,  showed  that  various  bacteria 
are  destroyed  within  a  short  time  by  the  fresh  blood  of  warm- 
hlnndcd  animals.  Thus  tlu>  anthrax  bacillus  in  rabbit's  blood  was 
usually  killed  in  from  two  to  four  hours  when  the  temperature  was 
maintained  at  37°-38°  C.,  and  the  same  result  was  obtained  with 
pigeon's  blood  at  41°  C.  But  when  the  blood  was  allowed  to  stand 
for  a  considerable  time,  or  was  heated  for  forty-five  minutes  to 
45°  C.,  it  served  as  a  culture  fluid,  and  an  abundant  development  of 
anthrax  bacilli  occurred  in  it.  Bacillus  subtilis  and  Bacillus  mega- 


ACTION   OF   BLOOD    SERUM    AND    OTHER    ORGANIC   LIQUIDS.     205 

therium  were  also  destroyed  in  two  hours  by  fresh  rabbit's  blood, 
but  it  was  without  action  on  Staphylococcus  pyogenes  aureus,  which 
at  a  temperature  of  37. 5°  C.  was  found  to  have  increased  in  num- 
bers at  the  end  of  two  hours.  Further  researches  by  Nissen  and 
Behring  show  that  there  is  a  wide  difference  in  the  blood  of  dif- 
ferent animals  as  to  germicidal  power,  and  that  certain  bacteria 
are  promptly  destroyed,  while  other  species  are  simply  restrained  for 
a  time  in  their  development  or  are  not  affected.  Thus  Nissen  found 
that  the  cholera  spirillum,  the  bacillus  of  anthrax,  the  bacillus  of 
typhoid  fever,  and  Friedlander's  pneumococcus  were  killed,  while 
Staphylococcus  pyogenes  aureus  and  albus,  the  streptococcus  of  ery- 
sipelas, the  bacillus  of  fowl  cholera,  the  bacillus  of  rothlauf,  and 
Proteus  hominis  were  able  to  multiply  in  rabbit's  blood  after  having 
been  restrained  for  a  short  time  in  their  development.  In  the  case 
of  the  cholera  spirillum  a  period  of  ten  to  forty  minutes  sufficed  for 
the  complete  destruction  of  a  limited  number,  but  when  the  number 
exceeded  1,200,000  per  cubic  centimetre  they  were  no  longer  de- 
stroyed with  certainty,  and  after  five  hours  an  increase  occurred. 
The  anthrax  bacillus  was  commonly  destroyed  within  twenty  minutes 
and  the  typhoid  bacillus  at  the  end  of  two  hours.  In  the  experi- 
ments of  Behriiig  and  Mssen  it  was  found  that  the  most  pronounced 
germicidal  effect  upon  the  anthrax  bacillus  was  obtained  from  the 
blood  of  the  rat,  an  animal  which  has  a  natural  immunity  against 
anthrax ;  while  the  blood  of  the  guinea-pig,  a  very  susceptible  ani- 
mal, had  no  restraining  effect  and  served  as  a  favorable  culture 
medium  for  the  anthrax  bacillus.  And  the  remarkable  fact  was  de- 
monstrated that  when  the  blood  of  a  rat  was  added  to  the  blood  of 
the  guinea-pig  in  the  proportion  of  1:8,  it  exercised  a  decided  re- 
straining influence  upon  the  growth  of  the  anthrax  bacillus.  Later 
researches  have  shown  that  cultivation  in  the  blood  of  an  immune 
animal  causes  an  attenuation  of  the  virulence  of  an  anthrax  cul- 
ture (Ogata  and  Jasuhara)  ;  and  also  that  the  injection  of  the  blood 
of  a  frog  or  rat — naturally  immune — into  a  susceptible  animal  which 
has  been  inoculated  with  a  virulent  culture  of  the  anthrax  bacillus, 
will  prevent  the  death  of  the  inoculated  animal. 

Buchner  has  shown  that  the  germicidal  power  of  the  blood  of 
dogs  and  rabbits  does  not  depend  upon  the  presence  of  the  cellular 
elements,  but  is  present  in  clear  serum  which  has  been  allowed  to 
separate  from  the  clot  in  a  cool  place.  Exposure  for  an  hour  to  a 
temperature  of  55°  C.  destroys  the  germicidal  action  of  serum  as 
well  as  of  blood  ;  the  same  effect  is  produced  by  heating  to  52°  C.  for 
six  hours  or  to  45.  C°  C.  for  twenty  hours.  The  germicidal  power 
of  blood  serum  is  not  destroyed  by  freezing  and  thawing,  but  is 
lost  after  it  has  been  kept  for  some  time.  Buchner 's  experiments  led 


206    ACTION   OF  BLOOD   SERUM   AND    OTHER   ORGANIC   LIQUIDS. 

him  to  the  conclusion  that  the  germicidal  power  of  fresh  blood 
serum  depends  upon  the  presence  of  some  albuminous  body  present 
in  it.  This  view  is  sustained  by  the  researches  of  Ogata,  who  has 
obtained  from  the  blood  of  dogs  and  other  animals  a  glycerin  ex- 
tract of  a  "  ferment"  which  is  insoluble  in  alcohol  or  in  ether  and 
which  has  germicidal  properties. 

According  to  Emmerich  and  Tsuboi  (1893),  when  the  serum- 
albumin  is  precipitated  by  alcohol,  dried  in  a  vacuum  at  40°  C.,  and 
dissolved  in  water  it  has  no  longer  any  germicidal  activity.  But  if 
the  precipitated  and  dried  albumin  is  dissolved  at  39°  C.  in  a  weak 
solution  (0.05-0.08  per  cent)  of  soda  or  potash  it  recovers  its  original 
germicidal  value. 

It  has  been  demonstrated  by  several  experimenters  that  other 
albuminous  fluids  possess  a  similar  germicidal  power.  Thus  Nuttall 
found  that  a  pleuritic  exudation  from  man  destroyed  the  anthrax 
bacillus  in  an  hour,  the  aqueous  humor  of  a  rabbit  in  two  hours. 
Wurz  has  experimented  with  fresh  egg  albumin,  and  found  that  the 
anthrax  bacillus  failed  to  grow  after  having  been  exposed  for  an  hour 
to  the  action  of  albumin  from  a  hen's  egg ;  other  bacteria  tested 
were  not  killed  so  promptly,  but  a  decided  germicidal  action  was 
manifested.  Prudden  has  shown  that  the  albuminous  fluid  obtained 
from  a  hydrocele,  or  from  the  abdominal  cavity  in  ascites,  possesses 
similar  germicidal  power  ;  and  Fokker  has  demonstrated  that  fresh 
in  ilk  destroys  the  vitality  of  certain  bacteria  which  induce  an  acid 
fermentation  of  this  fluid. 

The  results  heretofore  referred  to  induced  Hankin  to  experiment 
with  cell  globulin  obtained  from  the  spleen  or  lymphatic  glands  of  a 
dog  or  cat.  This  is  extracted  by  means  of  a  solution  of  chloride  of 
sodium,  the  solution  is  filtered,  and  the  globulin  precipitated  by  the 
;i«l<lition  of  alcohol.  The  precipitate  is  washed  and  again  dissolved 
in  salt  solution.  The  result  showed  that  this  cell  globulin  possesses 
germicidal  power  similar  to  that  of  blood  serum. 

Mucus.—  The  experiments  of  Wurtz  and  Lermoyez  (1893)  show 
that  nasal  mucus  has  germicidal  properties,  especially  for  the  anthrax 
bacilhls.  Walthard  (18!)3),  in  experiments  with  mucus  from  the  cer- 
vix uteri,  was  not  able  to  demonstrate  any  germicidal  action,  but 
arrived  at  the  conclusion  that  it  prevents  the  development  of  bacteria 
simply  because  it  is  an  unfavorable  medium.  Various  bacteria  were 
planted  upon  the  surface  of  cervical  mucus  in  Petri  dishes,  and  placed 
in  the  incubating  oven,  but  all  failed  to  grow. 

Nucleins  from  animal  and  vegetable  cells  have  been  shown  by 
Professor  Vaughau  and  his  associates  (1893)  to  possess  considerable 
germicidal  power.  The  nucleins  of  animal  origin  were  obtained  from 
the  testes  of  dogs  and  rats.  Dissolved  in  a  0.5-per-cent  solution  of 


ACTION   OF  BLOOD    SERUM  AND   OTHER   ORGANIC  LIQUIDS.      207 

caustic  potash  and  then  diluted  with  four  volumes  of  physiologic  salt 
solution  the  germicidal  activity  was  shown  by  the  facts  that  Staphylo- 
coccus  pyogenes  aureus,  and  the  anthrax  bacillus  without  spores, 
failed  to  grow  after  twenty  minutes'  exposure.  Kossel  (1893)  has 
obtained  similar  results  with  nucleins  from  the  thymus  gland  of  the 
calf. 

Urine. — The  experiments  of  Lehniann  show  that  fresh  urine  has 
a  decided  germicidal  power  for  the  cholera  spirillum  and  the  anthrax 
bacillus,  and  no  doubt  for  other  bacteria  as  well.  To  what  constitu- 
ent of  the  urine  this  is  due  has  not  been  determined,  but  it  may  be 
due  to  the  uric  acid  present. 


XIII. 
PRACTICAL  DIRECTIONS  FOR  DISINFECTION. 

THE  Committee  on  Disinfectants  of  the  American  Public  Health 
Association  (appointed  in  1884),  after  an  extended  investigation  with 
reference  to  the  germicidal  value  of  various  agents,  in  a  final  report 
submitted  in  1887  submits  the  following  "  Conclusions  ": 

The  experimental  evidence  recorded  in  this  report  seems  to  justify  the 
following  conclusions : 

The  most  useful  agents  for  the  destruction  of  spore-containing  infectious 
material  are — 

1.  Fire.    Complete  destruction  by  burning. 

2.  Steam  underpressure.    105°  C.  (221°  F.)  for  ten  minutes. 

3.  Boiling  in  water  for  half  an  hour. 

4.  Chloride  of  lime.1    A  four-per-cent  solution. 

5.  Mercuric  chloride.    A  solution  of  1:  500. 

For  the  destruction  of  infectious  material  which  owes  its  infecting  power 
to  the  presence  of  microorganisms  not  containing  spores,  the  committee  rec- 
ommends— 

1 .  Fire.     Complete  destruction  by  burning. 

2.  Boiling  in  water  for  ten  minutes. 

3.  Dry  heat.     110°  C.  (230°  F  )  for  two  hours. 

4.  Chloride  of  lime.     A  two-per-cent  solution. 

5.  Solution  of  chlorinated  soda.9    A  ten-per-cent  solution. 

6.  Mercuric  chloride.     A  solution  of  1: 2,000. 

7.  Carbolic  acid.    A  five  per-cent  solution. 

8.  Sulphate  of  copper.     A  five-per-cent  solution. 
J>.  Chloride  of  zinc.    A  ten-per-cent  solution. 

lo.  Sulphur  dioxide.*  Exposure  for  twelve  hours  to  an  atmosphere  con- 
taining at  least  four  volumes  per  cent  of  this  gas  in  presence  of 
moisture. 

The  committee  would  make  the  following  recommendations  with  refe- 
iv  nee  to  the  practical  application  of  these  agents  for  disinfecting  purposes: 

FOR  EXCRETA. 

(a)  In  the  sick-room : 

1.  Chloride  of  lime  in  solution,  four  per  cent. 
In  the  absence  of  spores: 

2.  Carbolic  acid  in  solution,  five  per  cent. 
Sulphate  of  copper  in  solution,  five  per  cent. 

1  Should  contain  at  least  twenty-five  per  cent  of  available  chlorine. 
•  Should  contain  at  least  three  per  cent  of  available  chlorine. 
1  This  will  require  the  combustion  of  between  three  and  four  pounds  of  sulphur 
l»r  every  thousand  cubic  feet  of  air  space. 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION. 

(b)  In  privy  vaults : 

1.  Mercuric  chloride  in  solution,  1:  500.  * 

2.  Carbolic  acid  in  solution,  five  per  cent. 

(c)  For  the  disinfection  and  deodorization  of  the  surface  of  masses  of  or- 
ganic material  in  privy  vaults,  etc. : 

Chloride  of  lime  in  powder. 

FOR  CLOTHING,    BEDDING,   ETC. 

(a)  Soiled  underclothing-,  bed  linen,  etc. : 

1.  Destruction  by  fire,  if  of  little  value. 

2.  Boiling-  for  at  least  half  an  hour. 

3.  Immersion  in  a  solution  of  mercuric  chloride  of  the  strength  of 

1 : 2,000  for  four  hours. 

4.  Immersion  in  a  two-per-cent  solution  of  carbolic  acid  for  four  hours. 
(6)  Outer  garments  of  wool  or  silk,  and  similar  articles,  which  would  be 

injured  by  immersion  in  boiling  water  or  in  a  disinfecting  solution: 

1.  Exposure  in  a  suitable  apparatus  to  a  current  of  steam  for  ten  min- 

utes. 

2.  Exposure  to  dry  heat  at  a  temperature  of  110°  C.  (230°  F.)  for  two 

hours. 
(c)  Mattresses  and  blankets  soiled  by  the  discharges  of  the  sick : 

1.  Destruction  by  fire. 

2.  Exposure  to  superheated  steam,  105°  C.  (221°  F.),  for  ten  minutes. 

(Mattresses  to  have  the  cover  removed  or  freely  opened.) 

3.  Immersion  in  boiling  water  for  half  an  hour. 

FURNITURE  AND  ARTICLES  OF  WOOD,    LEATHER,    AND  PORCELAIN. 

Washing,  several  times  repeated,  with — 
1.  Solution  of  carbolic  acid,  two  per  cent. 

FOR  THE    PERSON. 

The  hands  and  general  surface  of  the  body  of  attendants  of  the  sick,  and 
of  convalescents,  should  be  washed  with — 

1.  Solution  of  chlorinated  soda  diluted  with  nine  parts  of  water,  1: 10. 

2.  Carbolic  acid,  two-per-cent  solution. 

3.  Mercuric  chloride,  1: 1,000. 

FOR  THE  DEAD. 

Envelop  the  body  in  a  sheet  thoroughly  saturated  with — 

1.  Chloride  of  lime  in  solution,  four  per  cent. 

2.  Mercuric  chloride  in  solution,  1 :  500. 

3.  Carbolic  acid  in  solution,  five  per  cent. 

FOR  THE  SICK-ROOM  AND  HOSPITAL  WARDS. 

(a)  While  occupied,  wash  all  surfaces  with— 

1.  Mercuric  chloride  in  solution,  1: 1,000. 

2.  Carbolic  acid  in  solution,  two  per  cent. 

(6)  When  vacated,  fumigate  with  sulphur  dioxide  for  twelve  hours,  burn- 
ing at  least  three  pounds  of  sulphur  for  every  thousand  cubic  feet  of  air 
space  in  the  room ;  then  wash  all  surfaces  with  one  of  the  above-mentioned 
disinfecting  solutions,  and  afterward  with  soap  and  hot  water;  finally  throw 
open  doors  and  windows,  and  ventilate  freely. 

1  The  addition  of  an  equal  quantity  of  potassium  permanganate  as  a  deodorant, 
and  to  give  color  to  the  solution,  is  to  be  recommended.  [The  writer  no  longer  in- 
dorses this  recommendation.  See  his  paper  on  "  The  Disinfection  of  Excreta,"  ap- 
pended.] 

14 


210  PRACTICAL  DIRECTIONS   FOR   DISINFECTION. 

FOR  MERCHANDISE  AND  THE  MAILS. 

The  disinfection  of  merchandise  and  of  the  mails  will  only  be  required 
under  exceptional  circumstances ;  free  aeration  will  usually  be  sufficient.  If 
disinfection  seems  necessary,  fumigation  with  sulphur  dioxide  will  be  the 
only  practicable  method  of  accomplishing  it  without  injury. 

RAGS. 

(a)  Rags  which  have  been  usea  for  wiping  away  infectious  discharges 
should  at  once  be  burned. 

(b)  Rags  collected  for  the  paper-makers  during  the  prevalence  of  an  epi- 
demic should  be  disinfected,  before  they  are  compressed  in  bales,  by— 

1.  Exposure  to  superheated  steam  of  105°  C.  (221°  F.")  for  ten  minutes. 

2.  Immersion  in  boiling  water  for  half  an  hour. 

SHIPS. 

(a)  Infected  ships  at  sea  should  be  washed  in  every  accessible  place,  and 
especially  the  localities  occupied  by  the  sick,  with— 

1.  Solution  of  mercuric  chloride,  1 : 1,000. 

2.  Solution  of  carbolic  acid,  two  per  cent. 

The  bilge  should  be  disinfected  by  the  liberal  use  of  a  strong  solution  of 
mercuric  chloride. 

(b)  Upon  arrival  at  a  quarantine  station,  an   infected  ship  should  at 
once  be  fumigated  with  sulphurous  acid  gas,  using  three  pounds  of  sulphur 
for  every  thousand  cubic  feet  of  air  space;  the  cargo  should  then  be  dis- 
charged on  lighters;  a  liberal  supply  of  the  concentrated  solution  of  mercuric 
chloride  (four  ounces  to  the  gallon)  should  be  thrown  into  the  bilge,  and  at 
the  end  of  twenty- four  hours  the  bilge  watei  should  be  pumped  out  and  re- 
placed with  pure  sea  water;  this  should  be  repeated.    A  second  fumigation, 
after  the  removal  of  the  cargo,  is  recommended;  all  accessible  surfaces  should 
be  washed  with  one  of  the  disinfecting  solutions  heretofore  recommended, 
and  subsequently  with  soap  and  hot  water. 

FOR  RAILWAY  CARS. 

The  directions  given  for  the  disinfection  of  dwellings,  hospital  wards,  and 
ships  apply  as  well  to  infected  railway  cars.  The  treatment  of  excreta  with 
a  disinfectant,  before  they  are  scattered  along  the  tracks,  seems  desirable  at 
all  times  in  view  of  the  fact  that  they  may  contain  infectious  germs.  Dur- 
ing the  prevalence  of  an  epidemic  of  cholera  this  is  imperative.  For  this 
purpose  the  standard  solution  of  chloride  of  lime  is  recommended. 

DISINFECTION  BY   STEAM. 

The  Committee  on  Disinfectants,  in  tne  above-quoted  "Conclu- 
sions," recommends  the  use  of  "  steam  under  pressure,  105°  C.  (221° 
F.),  for  ten  minutes"  for  the  destruction  of  spore-containing  infec- 
tious material.  The  spores  of  all  known  pathogenic  bacteria  are  d<  '- 
stroyed  by  a  temperature  of  100°  C.  maintained  for  five  minutes,  and 
in  view  of  this  fact  the  temperature  fixed  by  the  committee  is  ample, 
and  to  exact  a  higher  temperature  or  longer  exposure  would  be  un- 
n-asonahle.  hut  in  practical  disinfection  the  temperature  required 
to  destroy  infectious  material  is  not  the  only  question  to  be  considered. 
Economy  in  the  construction  and  operation  of  the  steam  disinfecting 
apparatus  must  have  due  attention,  and  an  important  point  relates 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION.  211 

to  the  penetration  of  porous,  non-conducting  articles,  such  as  rolls  of 
blankets,  clothing,  etc.  These  points  have  been  the  subject  of  nu- 
merous experimental  investigations,  and  the  principles  involved 
have  been  elucidated,  especially  by  the  investigations  of  Esmarch 
(1887),  of  Budde  (1889),  and  of  Teuschner  (1890). 

It  has  been  shown  that  streaming  steam  is  more  effective  than 
confined  steam  at  the  same  temperature,  because  it  penetrates  porous 
objects  more  quickly.  Also  that  superheated,  "  dry  "  steam  is  not  as 
effective  as  flowing  steam  at  100°  C. ;  on  the  other  hand,  it  corre- 
sponds in  effectiveness  with  dry  air,  and  the  temperature  must  be 
raised  to  140°  to  150°  C.  in  order  to  quickly  destroy  the  spores  of 
bacilli. 

Esmarch's  investigations  show  that  streaming  steam  penetrates 
porous  objects,  like  rolled  blankets,  more  readily  than  confined 
steam ;  but  the  later  researches  of  Budde  and  of  Teuschner  show 
that  a  temperature  of  100°  C.  is  more  rapidly  reached  in  the  interior 
of  such  rolls  when  the  flowing  steam  is  under  pressure.  With  the 
same  pressure  (fifteen  pounds)  a  temperature  of  100°  C.  was  reached 
in  two  and  one-half  minutes  when  the  steam  was  flowing,  and  in 
eleven  minutes  by  steam  at  rest  (Budde).  Intermittent  pressure 
was  not  found  by  Budde  to  present  any  advantages  over  continuously 
flowing  steam  ;  on  the  contrary,  the  time  of  penetration  was  longer. 

Teuschner,  whose  investigations  are  the  most  recent,  arrives  at 
the  following  conclusions  : 

1.  Strongly  superheated  steam  is  not  to  be  recommended  for  practical 
disinfection.     On  the  contrary,  a  slight  superheating  of  the  steam,  such  as 
occurs  in  the  apparatus  of  Schimmel,  is  not  objectionable. 

2.  Those  forms  of  apparatus  in  which  the  steam  enters  from  above  are 
much  safer  and  quicker  in  their  disinfecting  action  than  those  in  which  this 
is  not  the  case.     In  the  construction  of  such  apparatus  care  must  be  taken, 
in  order  to  secure  penetration  of  the  objects,  that  the  air  and  steam  have  a 
free  escape  below. 

3.  Disinfection  is  hastened  by  previously  warming  the  apparatus. 

4.  The  most  rapid  disinfecting  action  is  secured  by  the  use  of  streaming 
steam  in  a  state  of  tension  (under  pressure). 

5.  Objects  which  have  been  in  contact  with  fatty  or    oily  substances 
require  a  longer  time  for  disinfection  than  those  which  have  not. 

6.  To  accomplish  disinfection  it  is  necessary  to  expel,  as  completely  as 
possible,  all  air  from  the  objects  to  be  disinfected,  and  also  to  secure  a  suffi- 
cient condensation  of  the  steam. 

7.  The  condensation  of  the  steam  advances  in  a  sharply  denned  line 
from  the  periphery  to  the  centre  of  porous  objects. 

8.  The  temperature  necessary  for  disinfection  is  only  found  in  the  zone 
where  condensation  has  already  taken  place. 

9.  Only  a  few  centimetres  from  the  zone  in  which  the  temperature  is 
100°  C.— when  disinfection  is  incomplete— there  may  be  places  in  which 
the  temperature  is  40°  C.  or  more  below  the  boiling  point. 


212  PRACTICAL   DIRECTIONS    FOR   DISINFECTION. 

DISINFECTION   OF   THE   HANDS. 

The  importance  of  a  reliable  method  of  disinfecting  the  hands  of 
surgeons,  obstetricians,  and  nurses  after  they  have  been  in  contact 
with  infectious  material  from  wounds,  puerperal  discharges,  etc.,  is 
now  fully  recognized,  and  some  surgeons  consider  it  necessary  to 
completely  sterilize  the  hands  before  undertaking  any  surgical  opera- 
tion which  will  bring  them  in  contact  with  the  freshly-cut  tissues. 
The  numerous  experiments  which  have  been  made  with  a  view  to 
ascertaining  the  best  method  of  accomplishing  such  sterilization  of 
the  hands  show  that  it  is  by  no  means  a  simple  matter  to  effect  it, 
and  especially  to  insure  the  destruction  of  microorganisms  con- 
cealed beneath  the  finger  nails.  Fiirbringer,  in  an  extended  series 
of  experiments  (1888),  found  that  a  preliminary  cleansing  with  soap 
and  a  brush  was  even  more  important  than  the  degree  of  potency  of 
che  disinfecting  wash  subsequently  applied.  He  recommends  the 
following  procedure : 

1.  Remove  all  visible  dirt  from  beneath  and  around  the  nails. 

2.  Brush  the  spaces  beneath  the  nails  with  soap  and  hot  water 
for  a  minute. 

3.  Wash  for  a  minute  in  alcohol  (not  below  eighty  per  cent),  and, 
before  this  evaporates,  in  the  following  solution  : 

4.  Wash  thoroughly  for  a  minute  in  a  solution  containing  1  :  500 
of  mercuric  chloride  or  three  per  cent  of  carbolic  acid. 

Roux  and  Reynes  tested  the  above  method  of  Fiirbringer,  and 
found  that  it  gave  better  results  than  others  previously  proposed,  al- 
though not  always  entirely  successful  in  securing  complete  steriliza- 

tinll. 

Boll  has  recently  (1890)  reported  favorable  results  from  the  fol- 
lowing method  : 

1.  Cleanse  the  finder  nails  from  visible  dirt  with  knife  or  nail  scissors. 

2.  Brush  the  hands  for  three  minutes  with  hot  water  and  potash  soap. 

:;  Wash  for  half  a  minute  in  a  three-per-cent  solution  of  carbolic  acid, 
and  subsequently  in  a  1  : 2,000  solution  of  mercuric  chloride. 

4.  Rub  the  spaces  beneath  the  nails  and  around  their  margins  with  iodo- 
form  gauze  wet  in  a  ftve-per-cent  solution  of  carbolic  acid. 

Welch,  as  a  result  of  extended  experiments  made  at  the  Johns 
Hopkins  Hospital,  recommends  the  following  procedure  : 

1.  The  nails  are  kept  short  and  clean. 

•-'  The  hands  are  washed  thoroughly  for  several  minutes  with  soap  and 
water,  the  water  being  as  warm  as  can  be  comfortably  borne,  and  being  fre- 
quently changed.  A  brush  sterilized  by  steam  is  used.  The  excess  of  soap 
is  washed  off  with  water. 

:*.  The  hands  are  immersed  for  one  or  two  minutes  in  a  warm  saturated 
solution  of  permanganate  of  notash  and  are  rubbed  over  thoroughly  with  a 
sterilized  swab. 


PRACTICAL   DIRECTIONS  FOR  DISINFECTION.  213 

4.  They  are   then  placed  in  a  warm  saturated  solution  of  oxalic  acid, 
where  they  remain  until  complete  decolorization  of  the    permanganate 
occurs. 

5.  They  are  then  washed  off  with  sterilized  salt  solution  or  water. 

6.  They  are  immersed  for  two  minutes  in  sublimate  solution,  1  :  500. 
The  bacteriological  examination  of  the  skin  thus  treated  yields  almost 

uniformly  negative  results,  the  material  for  the  cultures  being  taken  from 
underneath  and  around  the  nails.  This  is  the  procedure  now  employed  in 
the  gynecological  and  surgical  wards  of  the  hospital. 

THE   DISINFECTION   OF   EXCRETA. 

'  The  following  paper  by  the  present  writer  was  read  before  the 
Section  on  State  Medicine  at  the  last  (1891)  meeting  of  the  American 
Medical  Association  : 

The  Committee  on  Disinfectants  appointed  by  the  American  Public 
Health  Association  in  1884,  in  its  final  report  submitted  in  1887,  gives  the 
following  general  directions : 

"Disinfection  of  Excreta,  etc. — The  infectious  character  of  the  dejections 
of  patients  suffering  from  cholera  and  from  typhoid  fever  is  well  established, 
and  this  is  true  of  mild  cases  and  of  the  earliest  stages  of  these  diseases  as 
well  as  of  severe  and  fatal  cases.  It  is  probable  that  epidemic  dysentery, 
tuberculosis,  and  perhaps  diphtheria,  yellow  fever,  scarlet  fever,  and  typhus 
fever,  may  also  be  transmitted  by  means  of  the  alvine  discharges  of  the 
sick.  It  is,  therefore,  of  the  first  importance  that  these  should  be  disin- 
fected. In  cholera,  diphtheria,  yellow  fever,  and  scarlet  fever  all  vomited 
material  should  also  be  looked  upon  as  infectious.  And  in  tuberculosis, 
diphtheria,  scarlet  fever,  and  infectious  pneumonia  the  sputa  of  the  sick 
should  be  disinfected  or  destroyed  by  fire.  It  seems  advisable  also  to  treat 
the  urine  of  patients  sick  with  an  infectious  disease  with  one  of  the  disinfect- 
ing solutions  below  recommended. 

"Chloride  of  lime,  or  bleaching  powder,  is  perhaps  entitled  to  the  first 
place  for  disinfecting  excreta,  on  account  of  the  rapidity  of  its  action. 

44  The  following  standard  solution  is  recommended: 

"Dissolve  chloride  of  lime  of  the  best  quality, !  in  pure  water,  in  the  pro- 
portion of  six  ounces  to  one  gallon.  Use  one  quart  of  this  solution  for  the 
disinfection  of  each  discharge  in  cholera,  typhoid  fever,  etc.3  Mix  well  and 
leave  in  the  vessel  for  at  least  one  hour  before  throwing  into  the  privy  vault 
or  water  closet. 

"  The  same  directions  apply  to  the  disinfection  of  vomited  matters.  In- 
fected sputum  should  be  discharged  directly  into  a  cup  half-full  of  the  solu- 
tion. A  five-per-cent  solution  of  carbolic  acid  may  be  used  instead  of  the 
chloride  of  lime  solution,  the  time  of  exposure  to  the  action  of  the  disinfect- 
ant being  four  hours"  (op.  cit.,  pp.  237,  238). 

The  object  of  this  paper  is  to  inquire  whether  these  recommendations, 
which  were  based  upon  the  experimental  data  available  at  the  time  they 
were  made,  are  sustained  by  subsequent  investigations ;  and  whether  any 
other  agents  have  been  shown  to  possess  superior  advantages  for  the  pur- 
pose in  view. 

But  first  we  desire  to  call  attention  to  another  portion  of  the  report  of  the 

1  Good  chloride  of  lime  should  contain  at  least  twenty-five  per  cent  of  available 
chlorine  (page  92).  It  may  be  purchased  by  the  quantity  at  three  and  one-half  cents 
per  pound.  The  cost  of  the  standard  solution  recommended  if  therefore  but  little 
more  than  one  cent  a  gallon.  A  clear  solution  may  be  obtained  by  filtration  or  by 
decantation,  but  the  insoluble  sediment  does  no  harm  and  this  is  an  unnecessary  re- 
finement. 

8  For  a  very  copious  discharge  use  a  larger  quantity. 


214  PRACTICAL  DIRECTIONS  FOR  DISINFECTION. 

Committee  on  Disinfectants.     On  page  236  the  following  definition  of  disin 
fection  and  disinfectants  is  given : 

"  The  object  of  disinfection  is  to  prevent  the  extension  of  infectious  dis- 
eases by  destroying  the  specific  infectious  material  which  gives  rise  to  them. 
This  is  accomplished  by  the  use  of  disinfectants.  There  can  be  no  partial 
disinfection  of  such  material;  either  its  infecting  power  is  destroyed  or  it  is 
not.  In  the  latter  case  there  is  a  failure  to  disinfect.  Nor  can  there  be  any 
disinfection  in  the  absence  of  infectious  material." 

I  have  italicized  the  last  sentence  because  I  wish  to  call  especial  attention 
to  it.  I  am  frequently  asked,  "  What  is  the  best  disinfectant  to  put  into  a 
water  closet? "  Now,  if  a  closet  or  privy  vault  is  resorted  to  only  by  healthy 
pers  ons  and  no  infectious  material  has  been  thrown  into  it,  there  is  nothing 
111  it  to  disinfect,  and  the  recommendation  of  the  Committee  on  Disinfect- 
ants does  not  apply  to  it  at  all.  It  may  smell  badly,  and  in  this  case  the 
bad  odor  may  be  neutralized  by  the  use  of  deodorants ;  or  we  may  prevent 
the  putrefactive  decomposition  of  its  contents,  and  thus  prevent  the  forma- 
tion of  the  offensive  gases  given  off  as  a  result  of  such  decomposition,  by 
the  use  of  antiseptics.  But  to  accomplish  this  it  is  not  necessary  to  sterilize 
the  entire  contents  by  the  use  of  active  germicidal  agents. 

A  solution  of  sulphate  of  iron  or  of  chloride  of  zinc  is  a  useful  antiseptic 
and  deodorizing  agent,  and  the  Committee  on  Disinfectants,  in  making  its 
recommendations,  did  not  intend  to  discourage  the  use  of  such  agents.  But 
exact  experimental  data  showed  that  these  agents  could  not  be  depended 
upon  for  the  destruction  of  infectious  disease  germs,  and  the  recommenda- 
tions made  related  to  disinfection  in  the  strict  and  proper  use  of  the  term  as 
above  defined.  This  definition  is  now  accepted  by  sanitarians  in  all  parts 
of  the  world,  but  many  practising  physicians  still  use  the  term  disinfectant 
as  synonymous  with  deodorant.  For  example,  I  find  in  a  recent  sanitary 
periodical,  under  the  heading  "Medical  Excerpt,''  an  item  copied  from  the 
American  Journal  of  Obstetrics,  to  which  the  name  of  a  distinguished  gy- 
necologist is  attached,  in  which  the  following  statement  is  made  with  reference 
to  a  much-advertised  so-called  "disinfectant":  "Asa  disinfectant  I  have 
used  it  in  my  house  for  over  a  year  with  great  satisfaction."  Now,  the  agent 
referred  to  has  been  proved  by  exact  experiments  to  have  comparatively 
little  disinfecting  power,  although  it  is  a  very  good  deodorant.  According 
to  our  definition,  "  the  object  of  disinfection  is  to  prevent  the  extension  of 
infectious  diseases  by  destroying  the  specific  infectious  material  which  gives 
rise  to  them."  Are  we  to  suppose  that  the  distinguished  gynecologist  above 
quoted  had  such  infectious  material  in  his  house  "for  over  a  year  "  at  the 
time  he  was  employing  "  with  great  satisfaction  "  the  agent  he  recommends? 
If  not,  the  term  was  improperly  employed,  for  "  there  can  be  no  disinfec- 
tion in  the  absence  of  infectious  material."  I  wish  to  emphasize  this  point, 
t>ecause  I  have  reason  to  believe  that,  in  the  army  at  least,  the  recommen- 
dation of  the  Committee  on  Disinfectants  has  led  to  the  substitution  of  chlo- 
ride of  lime  for  cheaper  deodorants  and  antiseptic  agents — and  especially  for 
sulphate  of  iron— in  latrines  which  are  frequented  only  by  healthy  persons 
and  consequently  need  no  disinfection.  The  amount  of  chloride  of  lime 
issued  from  the  Medical  Purveying  Depot  at  San  Francisco  during  the  past 
si\  months  for  use  at  military  posts  on  the  Pacific  coast  is  more  than 
double  the  amount  of  sulphate  of  iron ;  but  there  has  been  no  epidemic  of 
an  infectious  disease,  and  probably  comparatively  little  call  for  the  use  of  a 
disinfecting  agent  in  the  sick-room.  We  quote  again  from  the  report  of  the 
Committee  on  Disinfectants : 

"  In  the  sick-room  we  have  disease  germs  at  an  advantage,  for  we  know 
where  to  find  them  as  well  as  how  to  kill  them.  Having  this  knowledge, 
not  to  apply  it  would  be  criminal  negligence,  for  our  efforts  to  restrict  the 
extension  of  infectious  diseases  must  depend  largely  upon  the  proper  use  of 
disinfectants  in  the  sick-room"  (op.  cit,  p.  237). 

"  The  injurious  consequences  which  are  likely  to  result  from  such  mis- 
apprehension and  misuse  of  the  word  disinfectant  will  be  appreciated  when 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION.  215 

it  is  known  that  recent  researches  have  demonstrated  that  many  of  the 
agents  which  have  been  found  useful  as  deodorizers  or  as  antiseptics  are  en- 
tirely without  value  for  the  destruction  of  disease  germs. 

"  This  is  true,  for  example,  as  regards  the  sulphate  of  iron,  or  copperas,  a 
salt  which  has  been  extensively  used  with  the  idea  that  it  is  a  valuable  dis- 
infectant. As  a  matter  of  fact,  sulphate  of  iron  in  saturated  solution  does 
not  destroy  the  vitality  of  disease  germs,  or  the  infecting  power  of  material 
containing  them.  This  salt  is,  nevertheless,  a  very  valuable  antiseptic,  and 
its  low  price  makes  it  one  of  the  most  available  agents  for  the  arrest  of  putre- 
factive decomposition"  (op.  cit.,  p.  237). 

Chloride  of  lime  is  also  a  valuable  antiseptic  and  deodorant,  and  I  know 
of  110  objection  to  substituting  it  for  sulphate  of  iron  other  than  the  question 
of  cost.  The  first  cost  of  chloride  of  lime,  by  the  quantity,  is  about  double 
that  of  sulphate  of  iron,  but  practically  the  difference  is  much  greater,  be- 
cause it  is  necessary  to  preserve  the  chloride  of  lime  in  air-tight  packages. 
When  exposed  to  the  air  it  deteriorates  in  value  very  rapidly.  It  is,  there- 
fore, necessary  to  pack  it  in  air-tight  receptacles  which  will  not  be  injured 
by  the  corrosive  action  of  free  chlorine,  and  in  comparatively  small  quanti- 
ties so  that  the  contents  of  a  package  may  be  used  soon  after  it  is  opened. 

We  now  proceed  to  consider  the  experimental  data  relating  to  the  germi- 
cidal  value  of  chloride  of  lime. 

The  Committee  on  Disinfectants  gave  it  "the  first  place  for  disinfecting 
excreta,  on  account  of  the  rapidity  of  its  action."  This  recommendation  was 
upon  experimental  data  obtained  in  the  pathological  laboratory  of  the  Johns 
Hopkins  University,  under  the  writer's  direction,  and  is  sustained  by  more 
recent  experiments  made  in  Germany. 

The  experiments  of  Bolton,  made  for  the  Committee  on  Disinfectants  in 
1886,  j»-ave  the  following  results  :  The  time  of  exposure  being  two  hours,  the 
typhoid  bacillus  and  cholera  spirillum  in  bouillon  cultures  were  killed  by  a 
solution  containing  one  part  to  one  thousand  parts  of  water  (containing  0.03 
per  cent  of  available  chlorine).  Anthrax  spores  were  killed  in  the  same  time 
by  a  solution  containing  0.3  per  cent  of  available  chlorine.  Typhoid  faeces 
were  sterilized  by  a  two-per-cent  solution,  and  in  several  instances  by  a  one- 
lialf-per-cent  solution ;  but  some  resistant  spores  of  non-pathogenic  bacilli  sur- 
vived in  two  experiments  in  which  a  solution  of  1 : 100  was  used.  In  bouillon 
cultures  to  which  ten  per  cent  of  dried  egg  albumin  had  been  added  the 
typhoid  bacillus  was  destroyed  by  one-half  per  cent  (1 : 200). 

Nissen,  whose  experiments  were  made  in  Koch's  laboratory  in  1890,  found 
that  anthrax  spores  were  destroyed  in  thirty  minutes  by  a  five-per-cent 
solution,  and  in  seventy  minutes  by  a  one-per-cent  solution.  In  his  experi- 
ments the  typhoid  bacillus  and  the  cholera  spirillum  were  destroyed  with 
certainty  in  five  minutes  by  a  solution  containing  0.12  per  cent  (1:  833) ;  the 
anthrax  bacillus  in  one  minute  by  1 : 1, 000 ;  Staphylococcus  pyogenes  aureus  in 
one  minute  by  1 :  500.  Experiments  made  by  the  same  author  on  the  sterili- 
zation of  f;eces  showed  that  one  per  cent  could  be  relied  upon  to  destroy  the 
bacillus  of  typhoid  fever  and  the  spirillum  of  cholera  in  faeces  in  ten  min- 
utes. 

Carbolic  Acid — The  Committee  on  Disinfectants  says:  "  A  five-per-cent 
solution  of  carbolic  acid  may  be  used  instead  of  the  chloride  of  lime  solution, 
the  time  of  exposure  to  the  action  of  the  disinfectant  being  four  hours." 
This  recommendation  is  made  in  view  of  the  fact  that  in  those  diseases  in 
which  it  is  most  important  to  disinfect  the  excreta  the  specific  germ  does  not 
form  spores.  This  is  now  believed  to  be  true  of  the  typhoid  bacillus,  the 
spirillum  of  cholera,  the  bacillus  of  diphtheria,  the  bacillus  of  glanders,  and 
the  streptococcus  of  erysipelas ;  and  it  has  been  shown  by  exact  experiments 
that  all  of  these  pathogenic  bacteria  are  destroyed  in  two  hours  by  a  one-per- 
cent solution,  or  less,  of  this  agent. 

Spores  require  for  their  destruction  a  stronger  solution  and  a  longer  time. 
Koch  found  a  one-per-cent  solution  to  be  without  effect  on  anthrax  spores 
after  fifteen  days'  exposure ;  a  two-per-cent  solution  retarded  their  develop- 


216  PRACTICAL  DIRECTIONS   FOR   DISINFECTION. 

ment,  but  did  not  destroy  their  vitality  in  seven  days;  a  three-per  cent  olu- 
tion  was  effective  in  two  days.  According  to  Nocht,  at  a  temperature  of 
37.50°  C.  anthrax  spores  are  killed  by  a  five-per-cent  solution  in  three  hours. 

Carbolic  acid  possesses  the  advantage  of  not  being  neutralized  by  the  sub- 
stances found  in  excreta,  or  by  the  presence  of  albumin.  ThusBolton  found 
that  the  addition  of  ten  per  cent  of  dried  albumin  to  a  bouillon  culture  of 
the  typhoid  bacillus  did  not  materially  influence  the  result,  the  bacillus  be- 
ing destroyed  in  two  hours  by  a  one-per-cent  solution. 

This  agent,  then,  is  firmly  established  as  a  valuable  disinfectant  for  ex- 
creta, but  we  still  give  the  preference  to  the  standard  solution  of  chloride 
of  lime  of  the  Committee  on  Disinfectants  for  use  in  the  sick-room,  "on 
account  of  the  rapidity  of  its  action,"  and  also  on  account  of  its  compara- 
tive cheapness. 

At  the  International  Sanitary  Conference  at  Rome  (1885)  the  writer,  who 
was  associated  with  Dr.  Koch  on  the  Committee  on  Disinfectants,  presented 
the  claims  of  chloride  of  lime,  and  in  the  recommendations  of  the  commit- 
tee it  was  placed  beside  carbolic  acid  with  the  following  directions: 

"  Carbolic  acid  and  chloride  of  lime  are  to  be  used  in  aqueous  solution. 

"Weak  solutions,  carbolic  acid,  two  percent;  chloride  of  lime,  one  per 
cent. 

' '  Strong  solutions,  carbolic  acid,  five  per  cent ;  «  hloride  of  lime,  four  per 
cent." 

The  strong  solutions  were  to  be  used  for  the  disinfection  of  excreta. 

Creolin,  a  coal-tar  product,  which  is  a  syrupy;  dark-brown  fluid  with  the 
odor  of  tar,  has  during  the  past  three  years  received  much  attention  from 
the  German  bacteriologists.  It  is  probably  the  same  product  which  was 
tested  under  the  writer's  direction  for  the  Committee  on  Disinfectants,  in 
1885,  under  the  name  of  "Little's  soluble  phenyle."  It  stood  at  the  head 
of  the  **  Commercial  Disinfectants  "  tested.  The  experiments  made  in  Ger- 
many show  that  it  is  not  so  active  for  spores  as  carbolic  acid,  but  that  it 
very  promptly  kills  known  pathogenic  bacteria,  in  the  absence  of  spores,  in 
solutions  of  two  per  cent  or  less.  Eisenberg  found  that  a  solution  of  two 
per  cent  killed  all  test  organisms  within  fifteen  minutes.  Esmarch  found 
it  especially  fatal  to  the  cholera  spirillum,  which  was  killed  by  solutions  of 
1  : 1,000  in  ten  minutes.  The  typhoid  bacillus  showed  much  greater  resist- 
ing power— a  one-half-per-cent  solution  failed  after  ten  minutes'  exposure. 
The  pus  cocci  was  still  more  resistant.  Behring  has  shown  that  the  pre- 
sence of  albumin  greatly  diminishes  its  germicidal  power.  As  a  deodorant 
it  is  superior  to  carbolic  acid,  and  on  this  account  is  to  be  preferred  in  the 
sick-room.  A  recently  prepared  emulsion  may  be  used  to  disinfect  the  liquid 
excreta  of  cholera  or  typhoid  patients,  in  the  proportion  of  four  per  cent, 
two  hours'  time  being  allowed  for  the  action  of  the  disinfectant.  The  ex- 
periments of  Jager  upon  pure  cultures  of  the  tubercle  bacillus  attached  to 
silk  threads  were  successful  in  destroying  the  infecting  power  of  these  cul- 
tures, as  tested  by  inoculation  into  the  anterior  chamber  of  the  eye  of  a 
rabbit,  when  solutions  of  two  per  cent  were  used. 

The  value  of  this  agent  as  a  disinfectant  is  then  fully  established ;  as  to 
its  cost  in  comparison  with  the  agents  heretofore  mentioned  I  am  not  in- 
formed. 

Quicklime.— Experiments  made  in  Koch's  laboratory  in  1887  by  Libo- 
rius  led  him  to  place  a  high  value  upon  recently  burned  quicklime  as  a  dis- 
infectant. More  recent  experiments  by  Jager,  Kitasato,  Pfuhl,  and  others 
buve  shown  that,  this  ;i«n-nt  has  considerable  jrermicidal  power  in  the,  ab- 
sence of  spores,  and  that  the  value  which  has  long  been  placed  upon  it  for 
the  treatment  of  excrementitious  material  in  latrines,  etc.,  and  as  a  wash  for 
exposed  surfaces,  is  justified  by  the  results  of  exact  experiments  made  upon 
known  pathogenic  bacteria.  The  germicidal  power  of  lime  is  not  interfered 
with  by  the  presence  of  albuminous  materi.il,  but  is  neutralized  by  phos- 
]>h-ites,  carbonates,  and  other  bases,  and  by  carbonic  acid. 

In  the  writer's  experiments  a  saturated  aqueous  solution  of  calcium  oxide 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION.  217 

failed  to  kill  typhoid  bacilli  ;  but  when  suspended  in  water  in  the  proportion 
of  1  : 40  by  weight  this  bacillus  was  killed  at  the  end  of  two  hours.  Anthrax 
spores  were  not  killed  in  the  same  time  by  a  lime  wash  containing  twenty 
per  cent  by  weight  of  pure  calcium  oxide.  According  to  Kitasato,  the 
typhoid  bacillus  and  the  cholera  spirillum  in  bouillon  cultures  are  destroyed 
by  the  addition  of  one-tenth  per  cent  of  calcium  oxide.  Pfuhl  experimented 
upon  sterilized  faeces  to  which  pure  cultures  of  the  typhoid  bacillus  or 
cholera  spirillum  were  added.  The  liquid  discharges  of  patients  with  typhoid 
fever  or  diarrhoea  were  used  for  the  purpose.  He  found  that  sterilization 
was  effected  at  the  end  of  two  hours  by  adding  fragments  of  calcium  hydrate 
in  the  proportion  of  six  per  cent,  and  that  three  per  cent  was  effective  in  six 
hours.  When  a  milk  of  lime  was  used  which  could  be  thoroughly  mixed 
with  the  dejecta  the  result  was  still  more  favorable.  A  standard  preparation 
of  milk  of  lime  containing  twenty  per  cent  of  calcium  hydrate  killed  the 
typhoid  bacillus  and  the  cholera  spirillum  in  one  hour  when  added  to  liquid 
faeces  in  the  proportion  of  two  per  cent. 

The  experiments  with  this  agent  show  that  time  is  an  important  factor, 
and  that  much  longer  exposures,  as  well  as  stronger  solutions,  are  required 
to  destroy  pathogenic  bacteria  than  is  the  case  with  chloride  of  lime.  For 
this  reason  we  still  give  the  last-named  agent  the  preference  for  the  disinfec- 
tion of  excreta  in  the  sick-room.  But  in  latrines  the  time  required  to  accom- 
plish disinfection  is  of  less  importance,  and  we  are  disposed  to  give  recently 
burned  quicklime  the  first  place  for  the  disinfection  of  excreta  in  privy 
vaults  or  on  the  surface  of  the  ground.  It  may  be  applied  in  the  form  of 
milk  of  lime,  prepared  by  adding  gradually  eight  parts,  by  weight,  of  water 
to  one  part  of  calcium  hydrate.  This  must  be  freshly  prepared,  or  protected 
from  the  air  to  prevent  the  formation  of  the  inactive  carbonate  of  lime. 

According  to  Behring,  lime  has  about  the  same  germicidal  value  as  the 
other  caustic  alkalies,  and  destroys  the  cholera  spirillum  and  the  bacillus  of 
typhoid  fever,  of  diphtheria,  and  of  glanders  after  several  hours'  exposure, 
in  the  proportion  of  fifty  cubic  centimetres  normal-lauge  per  litre.  Wood 
ashes  or  lye  of  the  same  alkaline  strength  may  therefore  be  substituted  for 
quicklime. 

Finally,  it  must  not  be  forgotten  that  we  have  a  ready  means  of  disinfect- 
ing excreta  in  the  sick-room  or  its  vicinity  by  the  application  of  heat. 
Exact  experiments,  made  by  the  writer  and  others,  show  that  the  thermal 
death-point  of  the  following  pathogenic  bacteria,  and  of  the  kinds  of  virus 
mentioned  is  below  60°  C.  (140°  F.):  Spirillum  of  cholera,  bacillus  of  an- 
thrax, bacillus  of  typhoid  fever,  bacillus  of  diphtheria,  bacillus  of  glanders, 
diplococcus  of  pneumonia  (Micrococcus  Pasteuri),  streptococcus  of  erysipelas, 
staphylococci  of  pus,  micrococcus  of  gonorrhoea,  vaccine  virus,  sheep  pox 
virus,  hydrophobia  virus.  Ten  minutes'  exposure  to  the  temperature  men- 
tioned may  be  relied  upon  for  the  disinfection  of  material  containing  any  of 
these  pathogenic  organisms,  except  the  anthrax  bacillus  when  in  the  stage 
of  spore  formation.  The  use,  therefore,  of  boiling  water  in  the  proportion 
of  three  or  four  parts  to  one  part  of  the  material  to  be  disinfected  may  be 
safely  recommended  for  such  material.  Or,  better  still,  a  ten-per-cent  solu- 
tion of  sulphate  of  iron  or  of  chloride  of  zinc  at  the  boiling  point  may  be 
used  in  the  same  way  (three  parts  to  one).  This  will  have  a  higher  boiling 
point  than  water,  and  will  serve  at  the  same  time  as  a  deodorant.  During 
an  epidemic  of  cholera  or  typhoid  fever  such  a  solution  might  be  kept  boil- 
ing in  a  proper  receptacle  in  the  vicinity  of  hospital  wards  containing 
patients,  and  would  serve  to  conveniently,  promptly,  and  cheaply  disinfect 
all  excreta. 

For  the  disinfection  of  fseces  in  privy  vaults,  etc.,  Vincent  (1895) 
gives  the  first  place  to  sulphate  of  copper,  which  should  be  used  in  the 
proportion  of  eight  to  ten  grammes  per  litre  of  contents,  together 
with  an  equal  part,  by  weight,  of  sulphuric  acid. 


PKACTICAL    IHKKCTIONS    F()K    DISINFECTION. 


DISINFECTION   IN    DIPHTHERIA. 

At  the  meeting  of  the  Tenth  International  Medical  Congress  in 
Berlin  (1890)  Loffler  made  an  important  communication  upon  the 
measures  to  be  taken  to  prevent  the  spread  of  diphtheria.  His  con- 
el  usions  are  summarized  as  follows  : 

1.  The  cause  of  diphtheria  is  the  diphtheria  bacillus,  which  is  found  in  the 
secretions  of  the  affected  mucous  membrane.  , 

2.  With  this  secretion  it  is  distributed  outside  of  the  body  and  may  be 
deposited  upon  anything  in  the  vicinity  of  the  sick. 

3.  Those  sick  with  diphtheria  carry  about  bacilli  capable  of  infecting 
others  so  long  as  there  is  the  slightest  trace  of  diphtheritic  deposit,  and  even 
for  several  days  after  such  deposit  has  disappeared. 

4.  Those  sick  with  diphtheria  are  to  be  rigidly  isolated  so  long  as  the 
diphtheria  bacilli  are  present  in  their  secretions.     Children  who  have  been 
sick  with  diphtheria  should  be  kept  from  school  for  at  least  four  weeks. 

5.  The  diphtheria  bacilli  may  preserve  their  vitality  in  dried  fragments 
of  diphtheritic  membrane  for  four  or  five  months.     Therefore  all  objects 
which  may  have  been  exposed  to  contact  with  the  excretions  of  those  sick 
with  diphtheria,  such  as  linen,  bedclothing,  utensils,  clothing  of  nurses,  etc., 
should  be  disinfected  by  boiling  in  water  or  treated  with  steam  at  100°  C. 
In  the  same  way  the  rooms  occupied  by  diphtheria  patients  are  to  be  care- 
fully disinfected.     The  floors  should  be  repeatedly  scrubbed   with  hot  sub- 
limate solution  (1:1,000)  and  the  walls  rubbed  down  with  bread. 

The  recommendation  made  by  Loffler  with  reference  to  rubbing 
d«»\vn  the  walls  of  an  infected  apartment  with  bread  is  based  upon 
the  experiments  of  Esmarch  (1887),  as  a  result  of  which  he  arrived 
at  the  conclusion  that  this  is  the  most  reliable  method  of  removing 
1  i.-tcteria  attached  to  the  walls  of  an  apartment.  Fresh  bread  is  used, 
and,  after  having  been  used,  is  destroyed  by  burning.  We  judge 
that  this  method  would  be  especially  applicable  to  painted  surfaces 
or  to  walls  covered  with  paper.  For  plastered  walls  the  liberal  ap- 
plication of  lime  wash  is  probably  the  safest  method  of  disinfection. 

I  ;•  •«  cntly  the  use  of  the  vapors  of  formaldehyde  has  been  proposed  for  the 
disinfection  of  the  sick-room,  hospital  wards,  etc.  Miquel  (1894)  does  not 
think  favorably  of  this  agent  for  the  purpose  indicated,  although  it  is  a  very 
active  germicide  and  may  bo  used  with  advantage  to  disinfect  certain  articles 
wh i«-li  run  be  exposed  to  the  vapors  in  a  closed  receptacle,  and  which  would 
he  injured  l.y  exposure  to  steam.  Lehmann  (1893)  has  shown  that  articles 
of  leather,  wool,  or  silks  and  furs  may  be  disinfected  in  this  way  without  in- 
jury, hut  the  vapor  will  not  penetrate'  to  the  interior  of  bundles.'  Theauthor 
last  named  considers  it  especially  well  adapted  for  the  disinfection  of  hair- 
hnishi-s  and  combs.  The  disinfection  of  the  sick-room  and  its  contents  by 
means  of  ammonia  has  been  proposed  by  von  Rigler  (1893).  His  experiments 
led  him  to  the  conclusion  that  one  kilo  of  liquid  ammonia,  poured  into  shal- 
low dishes,  would  sutlice  for  the  disinfection  of  one  hundred  cubic  metres  of 
space,  including  hangings,  furniture,  etc.  The  carefully  conducted  experi- 
ments of  de  I<Y,  udenreich  ils«i:i)did  not  give  results  favorable  to  this  mode 
of  disinfection, 


PART  THIRD. 


PATHOGENIC   BACTERIA. 

I.  MODES  OF  ACTION.  II.  CHANNELS  OF  INFECTION.    III.  SUSCEPTIBILITY  AND 
IMMUNITY.    IV.  PYOGENIC  BACTERIA.    V.  BACTERIA  IN  CROUPOUS  PNEU- 
MONIA.   VI.  PATHOGENIC  MICROCOCCI  NOT  DESCRIBED  IN  SECTIONS  IV. 
AND  V.    VII.  THE  BACILLUS  OF  ANTHRAX.    VIII.  THE  BACILLUS 
OF  TYPHOID  FEVER.    IX.  BACTERIA  IN  DIPHTHERIA.    X.  BAC- 
TERIA IN  INFLUENZA.    XI.  BACILLI  IN  CHRONIC  INFECTIOUS 
DISEASES.    XII.  BACILLI  WHICH  PRODUCE  SEPTICAE- 
MIA IN  SUSCEPTIBLE  ANIMALS.  XIII.  PATHOGENIC 
AEROBIC  BACILLI  NOT  DESCRIBED  IN  PREVIOUS 
SECTIONS.  XIV.  PATHOGENIC  ANAEROBIC 
BACILLI.  XV.  PATHOGENIC  SPIRILLA. 
XVI.  BACTERIA  IN  INFEC- 
TIOUS DISEASES. 


PART    THIRD. 
PATHOGENIC    BACTERIA. 


I. 
MODES  OF  ACTION. 

MANY  of  the  saprophytic  bacteria  are  pathogenic  for  man,  or  for 
one  or  more  species  of  the  lower  animals,  when  by  accident  or  ex- 
perimental inoculation  they  obtain  access  to  the  body  ;  these  may  be 
designated  facultative  parasites.  Other  species  which,  for  a  time 
at  least,  are  able  to  lead  a  saprophytic  mode  of  life  have  their  nor- 
mal habitat  in  the  bodies  of  infected  animals,  in  which  they  produce 
specific  infectious  diseases.  To  this  class  belong  the  cholera  spirillum, 
the  anthrax  bacillus,  the  bacillus  of  typhoid  fever,  and  various  other 
microorganisms  which  are  the  cause  of  specific  infectious  diseases  in 
some  of  the  lower  animals.  These  we  may  speak  of  as  parasites 
and  facultative  saprophytes.  Still  others  are  strict  parasites  and 
do  not  find  the  conditions  for  their  development  outside  of  the  bodies 
of  the  animals  which  they  infest,  except  under  the  special  conditions 
in  which  bacteriologists  have  succeeded  in  cultivating  some  of  them. 
The  best  known  strict  parasites  are  the  tubercle  bacillus,  the  bacillus 
of  leprosy,  the  spirillum  of  relapsing  fever,  and  the  micrococcus  of 
gonorrhoea. 

There  can  be  but  little  doubt  that  even  the  strict  parasites,  at  some 
time  in  the  past,  were  also  saprophytes,  and  that  the  adaptation  to  a 
parasitic  mode  of  life  was  gradually  effected  under  the  laws  of  natural 
selection.  In  a  previous  chapter  (Section  III.,  Part  Second)  we  have 
referred  to  the  modifications  in  biological  characters  which  may 
occur  as  a  result  of  special  conditions  of  environment.  Thus  we  may 
obtain  non-chromogenic  varieties  of  species  which  usually  produce 
pigment,  or  non-pathogenic  varieties  of  bacteria  which  are  usually 
pathogenic.  There  is  also  evidence  that  the  tubercle  bacillus,  a  strict 


MODES   OF   ACTION. 

parasite,  may  be  so  modified,  by  cultivation  for  successive  genera- 
tions in  a  culture  medium  containing  glycerin,  that  it  will  finally 
grow  in  ordinary  beef  infusion,  thus  showing  a  tendency  to  adapt 
itself  to  a  saprophytic  mode  of  life. 

Some  of  the  saprophytic  bacteria  are  indirectly  pathogenic  by 
reason  of  their  power  to  multiply  in  articles  of  food,  such  as  milk, 
cheese,  fish,  sausage,  etc.,  and  there  produce  poisonous  ptomaines 
which,  when  these  articles  are  ingested,  give  rise  to  various  morbid 
symptoms,  such  as  vomiting,  gastric  and  intestinal  irritation,  fever, 
etc.  Or  similar  symptoms  may  result  from  the  multiplication  of 
bacteria  producing  toxic  ptomaines  in  the  alimentary  canal.  No 
doubt  gastric  and  intestinal  disorders  are  largely  due-  to  this  cause, 
and  may  be  induced  by  a  variety  of  saprophytic  bacteria  when  these 
establish  themselves  in  undue  numbers  in  any  portion  of  the  ali- 
mentary tract.  In  Asiatic  cholera  the  same  thing  occurs,  but  with 
more  fatal  results  from  the  introduction  of  the  East  Indian  cholera 
germ  discovered  by  Koch.  This  is  pathogenic  for  man,  because  it  is 
able  to  multiply  rapidly  in  the  human  intestine,  and  there  produces  a 
toxic  substance  which,  being  absorbed,  gives  rise  to  the  morbid  pheno- 
mena of  the  disease.  The  spirillum  itself  does  not  enter  the  blood  or 
invade  the  tissues,  except  to  a  limited  extent  in  the  mucous  coat  of 
the  intestine,  and  the  true  explanation  of  its  pathogenic  power  is  no 
doubt  that  which  has  been  given. 

Other  microorganisms  invade  the  tissues  and  multiply  in  cer- 
tain favorable  localities,  but  have  not  the  power  of  developing  in  the 
blood,  in  which  they  are  only  found  occasionally  and  in  very  small 
numbers  or  not  at  all.  Thus  the  typhoid  bacillus  locates  itself  in  the 
intestinal  glands,  in  the  spleen,  and  in  the  liver,  forming  colonies  of 
limited  extent,  and  evidently  not  finding  the  conditions  extremely 
favorable  for  its  growth,  inasmuch  as  it  does  not  take  complete  pos- 
session of  these  organs.  The  symptoms  which  result  from  its  pre- 
sence are  doubtless  partly  due  to  local  irritation,  disturbance  of  func- 
tion, and,  in  the  case  of  the  intestinal  glands,  necrotic  changes 
induced  by  it.  But  in  addition  to  this  its  pathogenic  action  depends 
upon  the  production  of  a  poisonous  ptomaine  which  has  been  isolated 
and  studied  by  the  German  chemist  Brieger  (typhotoxine). 

Certain  saprophytic  bacteria,  when  injected  beneath  the  skin  of  a 
susceptible  animal,  multiply  at  the  point  of  inoculation  and  invade 
the  surrounding  tissues,  giving  rise  in  some  instances  to  the  forma- 
tion of  a  local  abscess,  in  others  to  an  infiltration  of  the  tissues  with 
bloody  serum,  and  in  others  to  extensive  necrotic  changes.  These 
local  changes  are  due  not  simply  to  the  mechanical  presence  of  the 
microorganisms  which  induce  them,  but  to  chemical  products  evolved 
during  the  growth  of  these  pathogenic  bacteria.  Indeed,  their  patho- 


MODES   OF   ACTION.  223 

genie  power  evidently  depends,  in  some  instances  at  least,  upon  these 
toxic  products  of  their  growth,  by  which  the  vital  resisting  power  of 
the  tissues  is  overcome. 

Among  the  bacteria  which  in  this  way  produce  extensive  local 
inflammatory  and  necrotic  changes  are  certain  anaerobic  species 
found  in  the  soil  and  in  putrefying  material,  such  as  the  bacillus  of 
malignant  oedema  and  the  writer's  Bacillus  cadaveris.  The  bacillus 
of  symptomatic  anthrax,  an  infectious  disease  of  cattle,  acts  in  the 
same  way.  All  of  these  produce  toxic  substances  which  have  a  very 
pronounced  local  action  upon  the  tissues  invaded  by  them.  Other 
bacteria,  while  they  develop  chiefly  in  the  vicinity  of  the  point  of 
entrance — by  accident  or  by  inoculation — produce  a  potent  toxic  sub- 
stance which  gives  rise  to  general  symptoms  of  a  serious  character, 
such  as  tetanic  convulsions  (bacillus  of  tetanus)  or  intense  fever  and 
nervous  phenomena  (micrococcus  of  erysipelas).  Again,  the  local 
irritation  resulting  from  the  presence  of  parasitic  bacteria  may  pri- 
marily give  rise  to  the  formation  of  new  growths  having  alow  grade 
of  vitality,  which  later  may  undergo  necrotic  changes,  as  in  tubercu- 
losis, glanders,  and  leprosy.  In  this  case  constitutional  symptoms 
are  not  present,  or  are  of  a  mild  character  during  the  development 
of  these  new  formations,  which  apparently  result  from  the  local  ac- 
tion of  substances  eliminated  during  the  growth  of  the  parasite, 
rather  than  from  its  simple  presence.  This  is  an  inference  based 
upon  the  fact  that  non-living  particles,  or  even  living  parasites,  as  in 
trichinosis,  do  not  produce  similar  new  growths  composed  of  cells, 
but  become  encysted  in  a  fibrous  capsule. 

In  pneumonia  we  have  a  local  process  in  which  one  or  more  lobes 
of  the  lung  are  invaded  by  a  pathogenic  micrococcus  (Micrococcus 
pneumonise  crouposse)  which  induces  a  fibrinous  exudation  that  com- 
pletely fills  the  air  cells.  How  far  the  symptoms  of  the  disease  are 
due  to  the  local  inflammation  and  disturbance  of  function,  and  to 
what  extent  they  may  be  due  to  the  absorption  of  a  soluble  toxic 
substance  evolved  as  a  result  of  the  growth  of  the  micrococcus,  has 
not  been  determined.  But  the  mild  character  of  the  general  symp- 
toms when  a  limited  area  of  lung  tissue  is  involved  leads  to  the  in- 
ference that  the  pathogenic  power  of  this  particular  pathogenic 
microorganism  is  chiefly  exercised  locally. 

The  pus  cocci  and  various  other  saprophytic  bacteria,  when  intro- 
duced beneath  the  skin,  give  rise  to  the  formation  of  abscesses,  un- 
attended by  any  very  considerable  general  disturbance  ;  and  also  to 
secondary  purulent  accumulations — metastatic  abscesses. 

That  this  is  not  due  simply  to  their  mechanical  presence  is  shown 
by  the  fact  that  powdered  glass  and  other  inert  substances,  when 
thoroughly  sterilized,  do  not  give  rise  to  pus  formation  when  intro- 


224  MODES  OF  ACTION. 

duced  beneath  the  skin  or  injected  into  the  cavity  of  the  abdomen. 
On  the  other  hand,  it  has  been  demonstrated  by  the  experiments  of 
Grawitz,  De  Bary,  and  others  that  certain  chemical  substances 
which  act  as  local  irritants  when  brought  in  contact  with  the  tissues 
may  induce  pus  formation  quite  independently  of  microorganisms  : 
nitrate  of  silver,  oil  of  turpentine,  and  strong  liquor  ammonise  have 
been  shown  to  possess  this  power.  And  it  has  been  demonstrated  by 
the  recent  experiments  of  Buchner  that  sterilized  cultures  of  a  long 
list  of  different  bacteria — seventeen  species  tested — give  rise  to  sup- 
puration when  introduced  into  the  subcutaneous  tissues. 

Buchner  has  further  shown  that  this  property  of  inducing  pus  for- 
mation resides  in  the  dead  bacterial  cells  and  not  in  soluble  products 
present  in  the  cultures.  For  the  clear  fluid  obtained  by  passing 
these  sterilized  cultures  through  a  porcelain  filter  gave  a  negative  re- 
sult, while  the  bacteria  retained  by  the  filter,  although  no  longer 
capable  of  development,  having  been  killed  by  heat,  invariably 
caused  suppuration. 

Individuals  suffering  from  malnutrition  are  more  susceptible  to 
invasion  by  specific  disease  germs  or  by  the  common  pus  cocci 
than  are  those  in  vigorous  health.  Thus  the  sufferers  from  starva- 
tion, from  crowd  poisoning,  sewer-gas  poisoning,  etc.,  are  not  only 
liable  to  be  early  victims  during  the  prevalence  of  an  epidemic  dis- 
ease, but  are  very  subject  to  abscesses,  boils,  ulcers,  etc.  A  slight 
abrasion  in  such  an  individual,  inoculated  by  the  ever-present  pus 
cocci,  may  give  rise  to  an  obstinate  ulcer  or  a  phlegmonous  inflam- 
mation. 

In  the  same  way  some  of  the  ordinary  saprophytes,  which  usually 
have  no  pathogenic  power,  may  be  pathogenic  for  an  animal  whose 
strength  is  reduced  by  disease  or  injury.  Thus  necrotic  changes 
may  occur  in  injured  tissues,  or  in  those  which  have  a  deficient  blood 
Mipply — from  occlusion  of  an  artery,  for  example — due  to  the  presence 
of  putrefactive  bacteria  which  are  incapable  of  development  in  the 
circulation  of  a  healthy  animal  or  in  healthy  tissues.  We  may  also 
have  a,  progressive  gangrene,  due  to  infection  of  wounds  by  bacteria 
which  are  able  to  invade  healthy  tissues.  This  is  seen  in  the  so- 
< -a  1  Inl  hospital  gangrene,  which  is  undoubtedly  due  to  microorgan- 
i>ms,  although  the  species  concerned  in  its  production  has  not  been 
determined,  owing  to  the  fact  that  modern  bacteriologists  have  had 
few,  if  any,  opportunities  for  studying  it.  The  history  of  the  disease, 
i  t  s  rapid  extension  in  infected  surgical  wards,  the  extensive  slough- 
ing which  occurs  within  a  few  hours  in  previously  healthy  wounds, 
and  the  effect  of  deep  cauterization  by  the  hot  iron,  nitric  acid,  or 
hromino  in  arresting  the  progress  of  the  disease,  all  support  this  view 
of  its  etiology.  Whether  it  is  due  to  a  specific  pathogenic  micro- 


MODES   OF   ACTION.  225 

organism,  or  to  exceptional  pathogenic  power  acquired  by  some  one 
of  the  common  bacteria  which  infest  suppurating  wounds,  cannot  be 
determined  in  the  absence  of  exact  experiments  by  modern  methods. 
But  the  latter  view  has  seemed  to  the  writer  the  most  probably  cor- 
rect. There  are  many  facts  which  go  to  show  that  pathogenic  viru- 
lence may  be  increased  by  cultivation  in  animal  fluids,  and  where 
wounded  men  are  brought  together  under  unfavorable  sanitary  con- 
ditions, as  has  been  the  case  where  hospital  gangrene  has  made  its 
appearance,  it  may  be  that  some  common  saprophyte  acquires  the 
power  of  invading  the  exposed  tissues  instead  of  simply  feeding  upon 
the  secretions  which  bathe  its  surface. 

Koch  has  described  a  progressive  tissue  necrosis  in  mice,  due  to  a 
streptococcus,  which  he  first  obtained  by  inoculating  a  mouse  in  the 
ear  with  putrid  material.  The  morbid  process  is  entirely  local  and 
rapidly  progressive,  causing  a  fatal  termination  in  about  three  days, 
without  invasion  of  the  blood. 

In  diphtheritic  inflammations  of  mucous  membranes  we  have 
a  local  invasion  of  the  tissues  and  a  characteristic  plastic  exudation. 
In  true  diphtheria  the  local  inflammation  and  necrotic  changes  in 
the  invaded  tissues  are  not  sufficient  to  account  for  the  serious  gen- 
eral symptoms,  and  we  now  have  experimental  evidence  that  the 
diphtheria  bacillus  produces  a  very  potent  toxic  substance  to  which 
these  symptoms  are  no  doubt  largely  due.  The  diphtheria  bacillus 
of  Loftier  appears  to  be  the  cause  of  the  fatal  malady  which  goes 
by  this  name,  but  undoubtedly  other  microorganisms  may  be  con- 
cerned in  the  formation  of  diphtheritic  false  membranes.  In  cer- 
tain forms  of  diphtheria,  and  especially  when  it  occurs  as  a  com- 
plication of  scarlet  fever,  measles,  and  other  diseases,  the  Klebs- 
Loffler  bacillus  is  absent,  and  a  streptococcus,  which  appears  to  be 
identical  with  Streptococcus  pyogenes,  is  found  in  considerable  num- 
bers and  is  probably  the  cause  of  the  diphtheritic  inflammation. 
An  epidemic  of  diphtheria  occurring  among  calves  was  studied  by 
Loffler,  and  is  ascribed  by  him  to  his  Bacillus  diphtheria  vitulo- 
rum.  The  same  bacteriologist  has  shown  that  the  diphtheria  of 
chickens  and  of  pigeons  is  due  to  a  specific  bacillus  which  differs 
from  that  found  in  human  diphtheria,  and  which  he  calls  Bacillus 
diphtherias  columbrarum. 

Recently  Prof.  Welch  has  studied  the  histological  lesions  pro- 
duced by  filtered  cultures  of  the  diphtheria  bacillus.  Cultures  in 
glycerin-bouillon,  several  weeks  old,  were  filtered  through  porce- 
lain, and  the  sterile  filtrate  was  injected  beneath  the  skin  of  guinea- 
pigs.  One  cubic  centimetre  of  this  filtrate  was  injected  into  a  gui- 
nea-pig on  the  10th  of  December,  and  two  cubic  centimetres  more  on 
the  14th  of  the  same  month.  The  animal  succumbed  at  the  end  of 
15 


226  MODES   OF   ACTION. 

three  weeks  and  five  days  after  the  first  inoculation.  At  the  autopsy 
"the  lymphatic  glands  of  the  inguinal  and  axillary  regions  were 
found  to  be  enlarged  and  reddened;  the  cervical  glands  were  swollen 
and  the  thyroid  gland  was  greatly  congested.  There  was  a  consider- 
able excess  of  clear  fluid  in  the  peritoneal  cavity.  Both  layers  of  the 
peritoneum  were  reddened,  the  vessels  of  the  visceral  layer  being  es- 
pecially injected.  The  spleen  was  enlarged  to  double  the  average 
size;  it  was  mottled,  and  the  white  follicles  were  distinctly  outlined 
against  the  red  ground.  The  liver  was  dark  in  color  and  contained 
much  blood.  .  .  .  The  kidneys  were  congested  and  the  cut  surface 
was  cloudy.  .  .  .  The  pericardial  sac  was  distended  with  clear  se- 
rum. Under  the  epicardium  were  many  ecchymotic  spots.  The 
lungs  exhibited  areas  of  intense  congestion  or  actual  haemorrhage 
into  the  tissues.  .  .  .  The  histological  lesions  in  this  case  are  identi- 
cal with  those  observed  by  us  in  connection  with  the  inoculation  of 
the  living  organisms." 

To  what  extent  non-specific  catarrhal  inflammations  of  mucous 
membranes  are  caused  by  the  local  action  of  microorganisms  has 
not  been  determined,  but  in  gonorrhoea  the  proof  is  now  considered 
satisfactory  that  the  "  gonococcus  "  of  Neisser  is  the  cause  of  the 
intense  local  inflammation  and  purulent  discharge.  In  this  disease 
the  action  of  the  pathogenic  microorganism  seems  to  be  limited  to 
the  tissues  invaded  by  it,  as  there  is  no  general  systemic  disturbance 
indicating  the  absorption  of  a  toxic  ptomaine. 

Chronic  catarrhal  inflammations  appear,  in  some  cases  at  least, 
to  be  kept  up  by  the  presence  of  microorganisms,  which  are  always 
found  in  the  discharges  from  inflamed  mucous  surfaces. 

The  influence  of  microorganisms,  and  especially  of  the  pus  cocci, 
in  preventing  the  prompt  healing  of  wounds,  is  now  well  established. 
An  extensive  suppurating  wound  or  collection  of  pus,  especially  if 
putrefactive  bacteria  are  present,  causes  fever  and  nervous  symp 
toms,  due  to  the  absorption  of  toxic  products.  More  intense  general 
symptoms  result  from  the  presence  of  the  streptococcus  of  pus  than 
from  the  less  pathogenic  staphylococci ;  this  is  seen  in  erysipelatous 
inflammations  and  in  puerperal  metritis  due  to  the  presence  of  this 
micrococcus.  Like  the  other  pus  cocci,  the  Streptococcus  pyogenes 
does  not  usually  invade  the  blood,  but  when  introduced  into  the  sub- 
cutaneous tissues  it  induces  a  local  inflammator}^  process,  with  a  ten- 
dency to  pus  formation,  and  it  invades  the  neighboring  lymph  chan- 
nels, in  which  the  conditions  appear  to  be  especially  favorable  for  its 
multiplication. 

Kinally,  certain  pathogenic  bacteria,  when  introduced  into  the 
bodies  of  susceptible  animals,  quickly  invade  the  blood  and  multiply 
in  it.  In  so  doing  they  necessarily  interfere  with  its  physiological 


MODES   OF   ACTION.  227 

functions  by  appropriating  for  their  own  use  material  required  for 
the  nutrition  of  the  tissues ;  and  at  the  same  time  toxic  substances 
are  formed  which  play  an  important  part  in  the  production  of  the 
morbid  phenomena,  which  in  this  class  of  diseases  very  commonly 
lead  to  a  fatal  result.  The  pathogenic  bacteria  which  invade  the 
blood  may  also,  in  certain  cases,  give  rise  to  local  necrosis  and  dis- 
turbance of  function  in  various  organs  in  a  mechanical  way  by 
blocking  up  the  capillaries. 

The  invasion  of  the  blood  which  occurs  in  anthrax  and  in  vari- 
ous forms  of  septicaemia  in  the  lower  animals,  induced  by  subcuta- 
neous inoculation  with  pure  cultures  of  certain  pathogenic  bacteria, 
does  not  generally  immediately  follow  the  inoculation.  Usually  a 
considerable  local  development  first  occurs,  which  gives  rise  to  more 
or  less  inflammation  of  the  invaded  tissues,  and  very  commonly  to 
an  effusion  of  bloody  serum  in  which  the  pathogenic  microorganism 
is  found  in  great  numbers.  Even  in  susceptible  animals  the  blood 
seems  to  offer  a  certain  resistance  to  invasion,  which  is  overcome 
after  a  time  by  the  vast  number  of  the  parasitic  host  located  in  the 
vicinity  of  the  point  of  inoculation,  aided  probably  by  the  toxic  sub- 
stances developed  as  a  result  of  their  vital  activity. 

The  experiments  of  Cheyne  (1886)  seem  to  show  that  in  the  case 
of  very  pathogenic  species,  like  the  anthrax  bacillus  or  Koch's  bacil- 
lus of  mouse  septicaemia,  a  single  bacillus  introduced  subcutaneously 
may  produce  a  fatal  result  in  the  most  susceptible  animals,  while 
greater  numbers  are  required  in  those  which  are  less  susceptible. 
Thus  a  guinea-pig  succumbed  to  general  infection  after  being  inocu- 
lated subcutaneously  with  anthrax  blood  diluted  to  such  an  extent 
that,  by  estimation,  only  one  bacillus  was  present  in  the  fluid  in- 
jected ;  and  a  similar  result  in  mice  was  obtained  with  Bacillus 
murisepticus.  In  the  case  of  the  microbe  of  fowl  cholera  (Bacillus 
septicaemias  haemorrhagicae)  Cheyne  found  that  for  rabbits  the  fatal 
dose  is  300,000  or  more,  that  from  10,000  to  300,000  cause  a  local 
abscess,  and  that  less  than  10,000  produce  no  appreciable  effect. 
The  common  saprophyte  Proteus  vulgaris  was  found  to  be  patho- 
genic for  rabbits  when  injected  into  the  dorsal  muscles  in  sufficient 
numbers.  But,  according  to  the  estimates  made,  225,000,000  were 
required  to  cause  death,  while  with  doses  of  from  9,000,000  to  112,- 
000,000  a  local  abscess  was  produced,  and  less  than  9,000,000  gave 
an  entirely  negative  result. 

Secondary  infections  occurring  in  the  course  of  specific  infec- 
tious diseases  are  of  common  occurrence.  Thus  a  pneumonia  may 
be  developed  in  the  course  of  an  attack  of  measles  or  of  typhoid 
fever  ;  or  infection  by  the  common  pus  cocci  in  the  course  of  scarlet 
fever,  typhoid  fever,  mumps,  etc.,  may  give  rise  to  local  abscesses, 


228  MODES   OF  ACTION. 

to  endocarditis,  etc.  Again,  mixed  infection  may  be  induced  by 
injecting  simultaneously  into  susceptible  animals  two  species  of  path- 
ogenic bacteria. 

Bumm,  Bockhart,  and  others  have  reported  cases  of  mixed  gonor- 
rhoeal  infection  in  which  the.pyogenic  micrococci  gave  rise  to  ab- 
scesses in  the  glands  of  Bartholin,  to  cystitis,  parametritis,  or  to 
"  gonorrhceal  inflammation  "  of  the  knee  joint.  Babes  gives  numer- 
ous examples  of  mixed  infection  in  scarlet  fever  and  in  other  diseases 
of  childhood.  Anton  and  Fiitterer  have  studied  the  question  of 
secondary  infection  in  typhoid  fever.  Karlinski  has  reported  a  case 
of  secondary  infection  with  anthrax  in  a  case  of  typhoid  fever,  infec- 
tion occurring  by  way  of  the  intestine.  Many  other  examples  of 
secondary  or  mixed  infection  are  recorded  in  the  recent  literature  of 
bacteriology  and  clinical  medicine,  but  enough  has  been  said  to  call 
attention  to  the  importance  of  the  subject. 

The  researches  of  Romer,  Kanthack  (1892),  and  others  show  that 
the  injection  of  the  filtered  products  of  certain  bacteria  (Bacillus 
pyocyaneus,  Vibrio  Metchnikovi,  etc.)  produces  a  decided  leucocy- 
tosis  in  the  animals  experimented  upon.  And  a  similar  result,  prob- 
ably from  a  like  cause,  has  been  shown  by  recent  experiments  to 
occur  in  pneumonia  (Billings)  and  other  infectious  diseases. 

Certain  bacterial  products  have  been  shown  by  experiment  to  pro- 
duce fever  when  injected  into  the  circulation  or  beneath  the  skin  of 
lower  animals ;  others  produce  rapid  respiration,  dilatation  of  pupils, 
diarrhoea,  and  paralysis  or  convulsions  (typhotoxin  of  Brieger, 
methyl-guanidin,  etc.) ;  the  toxic  effects  of  some  are  immediate  and 
of  others  more  or  less  remote  (toxalbumin  of  diphtheria) ;  others  have 
a  primary  toxic  effect  which  is  followed  after  a  time  by  toxic  symp- 
toms of  a  different  order  (Pneumobacillus  liquefaciens  bovis). 


II. 

CHANNELS  OF  INFECTION. 

WE  have  abundant  evidence  that  susceptible  animals  may  be  in- 
fected by  the  injection  of  various  pathogenic  bacteria  beneath  the 
skin,  and  accidental  infection  through  an  open  tvound  or  abrasion 
of  the  skin  is  the  common  mode  of  infection  in  tetanus,  erysipelas, 
hospital  gangrene,  and  the  "  traumatic  infectious  diseases"  generally. 
Other  infectious  diseases,  like  anthrax  and  glanders,  are  frequently 
transmitted  in  the  same  way.  •  We  have  also  satisfactory  evidence 
that  tuberculosis  may  be  transmitted  to  man  by  the  accidental  inocu- 
lation of  an  open  wound ;  and  in  view  of  the  fact  that  susceptible 
animals  are  readily  infected  in  this  way,  it  would  be  strange  if  it 
were  otherwise. 

The  question  whether  infection  may  occur  through  the  unbroken 
skin  has  been  studied  by  several  bacteriologists  and  an  affirmative 
result  obtained.  Thus  Schimmelbusch  produced  pustules  upon  the 
thigh  in  two  young  persons  suffering  from  pyaemia  by  rubbing  upon 
the  surface  a  pure  culture  of  Staphylococcus  pyogenes  aureus  which 
he  had  obtained  from  the  pus  of  a  furuncle.  The  same  author  also 
succeeded  in  infecting  rabbits  and  guinea-pigs  with  anthrax,  and 
rabbits  with  rabbit  septicaemia,  by  rubbing  pure  cultures  upon 
the  uninjured  skin.  Similar  results  had  previously  been  reported 
by  Roth,  who  also  showed  that  infection  might  occur  through 
the  uninjured  mucous  membrane  of  the  nose.  Machnoff  also  suc- 
ceeded in  infecting  guinea-pigs  with  anthrax  through  the  unin- 
jured skin  of  the  back,  and,  as  a  result  of  subsequent  microscop- 
ical examination  of  stained  sections,  arrived  at  the  conclusion  that, 
the  principal  channel  through  which  infection  was  accomplished  was 
the  hair  follicles.  Braunschweig,  in  a  series  of  experiments  in  which 
he  introduced  various  pathogenic  bacteria  into  the  conjunctival  sac 
of  mice,  rabbits,  and  guinea-pigs,  obtained  a  negative  result  with  the 
anthrax  bacillus,  the  bacillus  of  mouse  septicaemia,  the  bacillus  of 
chicken  cholera,  and  Micrococcus  tetragenus;  but  the  bacillus  ob- 
tained by  Ribbert  from  the  intestinal  diphtheria  of  rabbits  gave  a 
positive  result  in  five  mice,  two  guinea-pigs,  and  a  rabbit. 


CHANNELS   OP   INFECTION. 

Infection  through  the  mucous  membrane  of  the  intestine  no 
doubt  occurs  in  certain  diseases.  This  is  believed  to  be  a  common 
mode  of  the  infection  of  sheep  and  cattle  with  anthrax,  and  probably 
also  in  the  infectious  disease  of  swine  known  as  hog  cholera.  The 
anthrax  bacillus  would  be  destroyed  by  the  acid  secretions  of  the 
stomach,  but  if  spores  are  present  in  food  ingested  they  will  reach 
the  intestine.  The  experiments  of  Korkunoff  do  not,  however,  sup- 
port the  view  that  infection  is  likely  to  occur  in  this  way.  In  a  series 
of  experiments  upon  white  mice  fed  with  bread  containing  a  quantity 
of  anthrax  spores  the  result  was  uniformly  negative,  but  exception- 
ally infection  occurred  in  rabbits.  The  same  author  obtained  posi- 
tive results  in  rabbits  fed  with  food  to  which  a  pure  culture  of  the 
bacillus  of  chicken  cholera  had  been  added. 

Buchner,  in  experiments  upon  mice  and  guinea-pigs  fed  with 
material  containing  anthrax  spores,  obtained  a  positive  result  in  four 
out  of  thirty-three  animals.  This  is  no  doubt  the  usual  mode  of  in- 
fection in  typhoid  fever  in  man. 

Infection  may  also  occur  through  the  mucous  membrane  of  the 
respiratory  organs.  This  has  been  demonstrated  by  several  bac- 
teriologists, and  especially  by  the  experiments  of  Buchner,  who 
mixed  dried  anthrax  spores  with  ly  cop  odium  powder  or  pulverized 
charcoal,  and  caused  mice  and  guinea-pigs  to  respire  an  atmosphere 
containing  this  powder  in  suspension.  In  a  series  of  sixty- six  experi- 
ments fifty  animals  died  of  anthrax,  nine  of  pneumonia,  and  seven 
survived.  That  infection  did  not  occur  through  the  mucous  mem- 
brane of  the  alimentary  canal  was  proved  by  comparative  experi- 
ments in  which  animals  were  fed  with  double  the  quantity  of  spores 
used  in  the  inhalation  experiments.  Out  of  thirty-three  animals  fed 
in  this  way  but  four  contracted  anthrax.  That  infection  occurred 
through  the  lungs  was  also  demonstrated  by  the  microscopical  ex- 
amination of  sections  and  by  culture  experiments,  which  showed  that 
the  lungs  were  extensively  invaded,  while  in  many  cases  the  spleen 
contained  no  bacilli.  Positive  results  were  also  obtained  with  cul- 
tures of  the  anthrax  bacillus  not  containing  spores,  which  the  ani- 
mals were  made  to  inhale  in  the  form  of  spray.  But  in  this  case  a 
considerable  quantity  was  required,  and  a  sero-fibrinous  pneumonia 
was  usually  produced  as  well  as  general  infection;  the  inhalation  of 
>  mall  quantities  gave  no  result.  Positive  results  in  rabbits  were  also 
•  >l>taim>d  by  causing  them  to  inhale  considerable  quantities  of  a  spray 
containing  the  bacillus  of  chicken  cholera. 

The  fact  that  large  quantities  of  a  liquid  culture  of  these  virulent 
bacilli  were  required  to  infect  very  susceptible  animals  by  way  of 
the  pulmonary  mucous  membrane,  and  that  Buchner  failed  to  cause 
tht»  infection  of  tlu»se  animals  with  small  quantities  of  a  pure  culture 


CHANNELS   OF   INFECTION.  231 

inhaled  in  the  form  of  spray,  indicates  that  this  is  not  a  common 
mode  of  infection  in  the  absence  of  spores.  This  view  receives 
further  support  from  the  experiments  of  Hildebrandt,  who  made 
tracheal  fistulse  in  three  rabbits,  and,  after  the  wound  had  entirely 
healed,  injected  into  the  trachea  of  each  a  pure  culture  of  the  anthrax 
bacillus,  which  was  proved  to  be  virulent  by  inoculation  in  mice  or 
guinea-pigs.  All  of  the  animals  remained  in  good  health.  On  the 
other  hand,  three  rabbits  which  received  in  the  same  way  a  pure  cul- 
ture of  the  bacillus  of  rabbit  septicaemia  died  as  a  result  of  general 
infection. 

That  man  may  be  infected  with  anthrax  by  way  of  the  respira- 
tory organs  seems  to  be  well  established.  In  England  the  disease 
known  as  "  wool-sorter's  disease"  results  from  infection  in  this  way 
among  workmen  engaged  in  sorting  wool,  which  is  liable  to  contain 
the  spores  of  the  anthrax  bacillus  when  obtained  from  the  skin  of  an 
animal  which  has  fallen  a  victim  to  this  disease.  That  infection 
occurs  through  the  lungs  is  shown  by  the  fact  that  these  organs  are 
first  involved,  the  disease  being,  in  fact,  a  pulmonic  anthrax. 

While  these  experiments  prove  the  possibility  of  infection  through 
the  respiratory  mucous  membrane,  other  experiments  made  by  Hil- 
debrandt show  that  under  ordinary  circumstances  bacteria  suspended 
in  the  air  do  not  reach  the  trachea  in  rabbits,  but  are  deposited  upon 
the  mucous  membrane  of  the  mouth,  nares,  and  fauces.  In  healthy 
rabbits  the  tracheal  mucus  was,  as  a  rule,  found  to  be  free  from  bac- 
teria, while  they  were  very  numerous  in  mucus  obtained  from  the 
mouth  or  nares.  But  when  a  rabbit  was  made  to  inhale  for  half  an 
hour  an  atmosphere  charged  with  the  spores  of  Aspergillus  f  umigatus 
their  presence  in  the  lungs  was  demonstrated  by  cultivation,  the  ani- 
mal being  killed  for  the  purpose  half  an  hour  after  the  inhalation 
experiment. 

The  rapidity  with  which  infection  may  occur  is  shown  by  the 
experiments  of  Nissen,  Pfuhl,  and  others.  In  mice  inoculated  with 
anthrax  bacilli  at  the  tip  of  the  tail  fatal  anthrax  has  resulted, 
although  the  tail  was  amputated  ten  minutes  after  the  inoculation. 
Schimmelbusch  inoculated  fresh  wounds  with  anthrax  cultures  (in 
mice)  and  immediately  after  treated  the  wounds  with  strong  anti- 
septic solutions,  but  the  animals  succumbed  to  infection.  Cultures 
of  the  anthrax  bacillus  have  been  obtained  from  the  liver,  spleen,  and 
kidneys  half  an  hour  after  the  infection  of  an  open  wound  on  the 
surface  of  the  body  (Schimmelbusch  and  Ricker).  The  experiments 
of  Sherrington  and  others  show  that  pathogenic  bacteria  may  escape 
by  way  of  the  kidneys  into  the  bladder,  or  through  the  liver  into  the 
gall  bladder.  But  his  experiments  indicate  that  such  escape  does  not 
occur  through  healthy  organs.  Non-pathogenic  bacteria  injected 


232  CHANNELS   OF   INFECTION. 

into  the  circulation  were  not  found  in  the  urine,  and  when  a  consid- 
erable quantity  of  a  pathogenic  species  was  injected  into  a  vein  there 
was  no  immediate  appearance  of  bacteria  in  the  urine,  but  they  were 
found  later,  probably  as  a  result  of  lesions  in  the  secreting  organ  due 
to  their  local  action  or  to  that  of  their  toxic  products.  In  man  the 
presence  of  pathogenic  bacteria  in  the  urine  has  been  frequently  veri- 
fied, especially  in  typhoid  fever,  pneumonia,  and  streptococcus  in- 
fection. When,  as  a  result  of  the  establishment  of  foci  of  infection 
in  the  liver,  localized  necrosis  of  tissue  occurs,  the  pathogenic  bac- 
teria to  which  the  infection  is  due  escape  with  the  bile  and  enter 
the  intestine.  It  is  probable  that  escape  through  the  walls  of  the 
intestine  does  not  occur  unless  there  is  a  local  lesion  of  some  kind,  as 
in  typhoid  fever. 

The  presence  of  tubercle  bacilli  in  the  milk  of  cows  has  been 
repeatedly  demonstrated,  and  in  a  certain  proportion  of  the  cases 
they  have  been  found  in  the  milk  of  cows  whose  udders  gave 
no  evidence  of  being  the  seat  of  a  tubercular  process.  Usually,  how- 
ever, when  tubercle  bacilli  are  found  in  the  milk  the  cow's  udder 
is  already  involved  in  the  disease.  The  milk  of  women  with  puer- 
peral fever  has  been  found  to  contain  streptococci ;  and  in  mastitis 
from  a  localized  infection  by  pyogenic  cocci  these  are  found  in  the 
milk.  It  must  be  remembered,  however,  that  both  Staphylococcus 
albus  and  aureus  have  been  found  in  the  milk  of  healthy  women. 
The  micrococcus  of  pneumonia  has  been  found  in  the  milk  of  women 
suffering  from  croupous  pneumonia  (Foa,  and  Bordoni-Uffreduzzi) . 
Various  observers  (Brunner,  Tizzoni,  von  Eiselsberg)  have  reported 
the  presence  of  pus  cocci  in  the  sweat  of  patients  suffering  from  sep- 
ticaemia, and  the  experiments  of  Brunner  indicate  that  they  may  have 
escaped  through  the  sweat  glands.  This,  however,  does  not  appear 
to  be  definitely  established. 


III. 

SUSCEPTIBILITY  AND   IMMUNITY. 

No  questions  in  general  biology  are  more  interesting,  or  more 
important  from  a  practical  point  of  view,  than  those  which  relate  to 
the  susceptibility  of  certain  animals  to  the  pathogenic  action  of  cer- 
tain species  of  bacteria,  and  the  immunity,  natural  or  acquired,  from 
such  pathogenic  action  which  is  possessed  by  other  animals.  It  has 
long  been  known  that  certain  infectious  diseases,  now  demonstrated 
to  be  of  bacterial  origin,  prevail  only  or  principally  among  animals 
of  a  single  species.  Thus  typhoid  fever,  cholera,  and  relapsing 
fever  are  diseases  of  man,  and  the  lower  animals  do  not  suffer  from 
them  when  they  are  prevailing  as  an  epidemic.  On  the  other  hand, 
man  has  a  natural  immunity  from  many  of  the  infectious  diseases  of 
the  lower  animals,  and  diseases  of  this  class  which  prevail  among 
animals  are  frequently  limited  to  a  single  species.  Again,  several 
species,  including  man,  may  be  susceptible  to  a  disease,  while  other 
animals  have  a  natural  immunity  from  it.  Thus  tuberculosis  is 
common  to  man,  to  cattle,  to  afpes,  and  to  the  small  herbivorous  ani- 
mals, while  the  carnivora  are,  as  a  rule,  immune  ;  anthrax  may  be 
communicated  by  inoculation  to  man,  to  cattle,  to  sheep,  to  guinea- 
pigs,  rabbits,  and  mice,  but  the  rat,  the  dog,  carnivorous  animals,  and 
birds  are  generally  immune  ;  glanders,  which  is  essentially  a  disease 
of  the  equine  genus,  may  be  communicated  to  man,  to  the  guinea- 
pig,  and  to  field  mice,  while  house  mice,  rabbits,  cattle,  and  swine 
are  to  a  great  extent  immune. 

In  addition  to  this  general  race  immunity  or  susceptibility  we 
have  individual  differences  in  susceptibility  or  resistance  to  the  ac- 
tion of  pathogenic  bacteria,  which  may  be  either  natural  or  acquired. 
As  a  rule,  young  animals  are  more  susceptible  than  older  ones. 
Thus  in  man  the  young  are  especially  susceptible  to  scarlet  fever, 
whooping  cough,  and  other  "children's  diseases,"  and  after  forty 
years  of  age  the  susceptibility  to  tubercular  infection  is  very  much 
diminished.  Among  the  lower  animals  it  is  a  matter  of  common 
laboratory  experience  that  the  very  young  of  a  susceptible  species 
may  be  infected  when  inoculated  with  an  "attenuated  culture" 
which  older  animals  of  the  same  species  are  able  to  resist. 


234  SUSCEPTIBILITY    AMD    IMMUNITY. 

Considerable  differences  as  to  susceptibility  may  also  exist  among 
adults  of  the  same  species.  In  man  these  differences  in  individual 
susceptibility  to  infectious  diseases  are  frequently  manifested.  Of  a 
number  of  persons  exposed  to  infection  in  the  same  way,  some  may 
. -scape  entirely  while  others  have  attacks  differing  in  severity  and 
duration.  In  our  experiments  upon  the  lower  animals  we  constantly 
meet  with  similar  results,  some  individuals  proving  to  be  exception- 
ally resistant.  Exceptional  susceptibility  or  immunity  may  be  to 
some  extent  a  family  characteristic  or  one  of  race.  Thus  the  negro 
race  is  decidedly  less  subject  to  yellow  fever  than  the  white  race, 
and  this  disease  is  more  fatal  among  the  fair-skinned  races  of  the 
north  of  Europe  than  among  the  Latin  races  living  in  tropical  or  sub- 
tropical regions.  On  the  other  hand,  small-pox  appears  to  be  excep- 
tionally fatal  among  negroes  and  dark-skinned  races  generally. 

A  very  remarkable  instance  of  race  immunity  is  that  of  Algerian 
sheep  against  anthrax,  a  disease  which  is  very  fatal  to  other  sheep. 

In  the  instances  mentioned  race  immunity  is  probably  an  ac- 
quired tolerance  due  to  natural  selection  and  inheritance.  If,  for 
example,  a  susceptible  population  is  exposed  to  the  ravages  of  small- 
pox, the  least  susceptible  individuals  will  survive  and  may  be  the  pa- 
rents of  children  who  will  be  likely  to  inherit  the  special  bodily  char- 
acters upon  which  this  comparative  immunity  depends.  The  ten- 
dency of  continuous  or  repeated  exposure  to  the  same  pathogenic 
agent  will  evidently  be  to  establish  a  race  tolerance  ;  and  there  is 
n '.ison  to  believe  that  such  has  been  the  effect  in  the  case  of  some 
of  the  more  common  infectious  diseases  of  man,  which  have  been 
noticed  to  prevail  with  especial  severity  when  first  introduced  among 
a  virgin  population,  as  in  the  islands  of  the  Pacific,  etc. 

In  the  same  way  we  may  explain  the  immunity  which  carnivor- 
ous animals  have  for  anthrax  and  various  forms  of  septicaemia  to 
which  the  herbivora  are  very  susceptible  when  the  pathogenic  germ 
is  introduced  into  their  bodies  by  inoculation.  From  time  immemo- 
rial the  carnivora  have  been  in  the  habit  of  fighting  over  the  dead 
bodies  of  herbivorous  animals,  some  of  which  may  have  fallen  a  prey 
to  these  infectious  germ  diseases,  and  in  their  fighting  they  receive 
wounds,  inoculated  with  the  infectious  material  from  these  bodies, 
which  would  be  fatal  to  a  susceptible  animal.  If  at  any  time  in  the 
j.ast  a  similar  susceptibility  existed  among  the  carnivora,  with  indi- 
vidual differences  as  to  resisting  power,  it  is  evident  that  there  would 
be  a  constant  tendency  for  the  most  susceptible  individuals  to  perish 
and  for  the  least  susceptible  to  survive. 

But  if  we  admit  this  to  be  a  probable  explanation  of  the  immu- 
nity of  carnivorous  animals  from  septic  infection,  we  have  not  yet 
t-\|.l aiiicd  tin-  praise  reason  for  the  immunity  enjoyed  by  the 


SUSCEPTIBILITY   AND    IMMUNITY.  235 

selected  individuals  and  their  progeny.  The  essential  difference  be- 
tween a  susceptible  and  immune  animal  depends  upon  the  fact  that 
in  one  the  pathogenic  germ,  when  introduced  by  accident  or  ex- 
perimental inoculation,  multiplies  and  invades  the  tissues  or  the 
blood,  where,  by  reason  of  its  nutritive  requirements  and  toxic  pro- 
ducts, it  produces  changes  in  the  tissues  and  fluids  of  the  body  incon- 
sistent with  the  vital  requirements  of  the  infected  animal ;  while  in 
the  immune  animal  multiplication  does  not  occur  or  is  restricted  to  a 
local  invasion  of  limited  extent,  and  in.  which  after  a  time  the  re- 
sources of  nature  suffice  to  destroy  the  parasitic  invader. 

Now  the  question  is,  upon  what  does  this  essential  difference  de- 
pend ?  Evidently  upon  conditions  favorable  or  unfavorable  to  the 
development  of  the  pathogenic  germ ;  or  upon  its  destruction  by 
some  active  agent  present  in  the  tissues  or  fluids  of  the  body  of  the 
immune  animal;  or  upon  a  neutralization  of  its  toxic  products  by  some 
substance  present  in  the  body  of  the  animal  which  survives  infec- 
tion. 

What,  then,  are  the  unfavorable  conditions  which  may  be  supposed 
to  prevent  development  in  immune  animals  ?  In  the  first  place,  the 
temperature  of  the  body  may  not  be  favorable.  Certain  pathogenic 
bacteria  are  only  able  to  develop  within  very  narrow  temperature  lim- 
its, and,  if  all  other  conditions  were  favorable,  could  not  be  expected 
to  multiply  in  the  bodies  of  cold-blooded  animals.  Or  the  temperature 
of  warm-blooded  animals,  and  especially  of  fowls,  may  be  above  the 
point  favorable  for  their  development.  This  is  the  explanation 
offered  by  Pasteur  of  the  immunity  of  fowls,  which  are  usually  re- 
fractory against  anthrax  ;  and  in  support  of  this  view  he  showed  by 
experiment  that  when  chickens  are  refrigerated  after  inoculation,  by 
being  partly  immersed  in  cold  water,  they  are  liable  to  become  in- 
fected and  to  perish.  But,  as  pointed  out  by  Koch,  the  sparrow, 
which  has  a  temperature  as  high  as  that  of  the  chicken,  may  con- 
tract anthrax  without  being  refrigerated.  We  must  not,  therefore, 
too  hastily  conclude  that  the  success  in  Pasteur's  experiment  de- 
pended alone  upon  a  reduction  of  the  body  heat.  Gibier  has  shown 
that  the  anthrax  bacillus  may  multiply  in  the  bodies  of  frogs  or 
fish,  if  these  are  kept  in  water  having  a  temperature  of  35°  C. 
But  the  anthrax  bacillus  grows  within  comparatively  wide  tempera- 
ture limits,  while  other  pathogenic  bacteria  are  known  to  have  a 
more  restricted  temperature  range  and  would  be  more  decidedly 
influenced  by  this  factor — e.g.,  the  tubercle  bacillus. 

The  composition  of  the  body  fluids,  and  especially  their  reaction, 
13  probably  a  determining  factor  in  some  instances.  Thus  Behring 
has  ascribed  the  failure  of  the  anthrax  bacillus  to  develop  in  the 
white  rat,  which  possesses  a  remarkable  immunity  against  anthrax, 


236  srsCKI'TIHILITY    AND    IMMUNITY. 

to  the  highly  alkaline  reaction  of  the  blood  and  tissue  juices  of  this 
animal.  Behring  claims  to  have  obtained  experimental  proof  of  the 
truth  of  this  explanation  by  feeding  white  rats  on  an  exclusively 
vegetable  diet  or  by  adding  acid  phosphate  of  lime  to  their  food,  by 
which  means  this  excessive  alkalinity  of  the  blood  is  diminished. 
Rats  so  treated  are  said  to  lose  their  natural  immunity,  and  to  die  as 
a  result  of  inoculation  with  virulent  cultures  of  the  anthrax  bacillus. 

The  experiments  of  Nuttall,  Behring,  Buchner,  and  others  have 
established  the  fact  that  recently  drawn  blood  of  various  animals 
possesses  decided  germicidal  power,  and  Buchner  has  shown  that 
this  property  belongs  to  the  fluid  part  of  the  blood  and  not  to  its 
cellular  elements.  It  has  also  been  shown  that  aqueous  humor,  the 
fluid  of  ascites,  and  lymph  from  the  dorsal  lymph  sac  of  a  frog 
possess  the  same  power.  This  power  to  kill  bacteria  is  destroyed  by 
heat,  and  is  lost  when  the  blood  has  been  kept  for  a  considerable 
time,  but  it  is  not  neutralized  by  freezing.  Further,  this  power  to 
destroy  bacteria  differs  greatly  for  different  species,  being  very  de- 
cided in  the  case  of  certain  pathogenic  bacteria,  less  so  for  others, 
and  absent  in  the  case  of  certain  common  saprophytes.  Behring 
has  also  shown  that  the  blood  of  different  animals  differs  consider- 
ably in  this  regard,  and  that  the  blood  of  the  rat  and  of  the  frog, 
which  animals  have  a  natural  immunity  against  anthrax,  is  espe- 
cially fatal  to  the  anthrax  bacillus.  The  experiments  made  show 
that  this  germicidal  power  is  very  prompt  in  its  action,  but  that  it  is 
limited  as  to  the  number  of  bacteria  which  can  be  destroyed  by  a 
given  quantity  of  blood  serum.  When  the  number  is  excessive,  de- 
velopment occurs  after  an  interval  during  which  a  limited  destruc- 
tion has  taken  place.  It  would  appear  that  the  element  in  the  blood 
to  which  this  germicidal  action  is  due  is  neutralized  in  exercising 
this  power  ;  and  as,  independently  of  this,  blood  serum  is  an  excel- 
lent culture  medium  for  bacteria,  an  abundant  development  takes 
place  when  the  destruction  has  been  incomplete. 

Buchner  (1880)  first  proved  by  experiment  that  the  germicidal 
power  of  the  blood  of  dogs  and  rabbits  does  not  depend  upon  the 
presence  of  the  cellular  elements,  but  is  present  in  clear  serum  which 
has  been  allowed  to  separate  from  the  clot  in  a  cool  place.  Exposure 
for  an  hour  to  a  temperature  of  55°  C.  destroys  the  germicidal  action 
of  serum  as  well  as  of  blood. 

The  researches  of  Buchner,  of  Hankin,  and  others,  show  that  this 
germicidal  power  of  fresh  blood  serum  depends  upon  the  presence  of 
proteids,  to  which  the  first-named  bacteriologist  has  given  the  name 
of  " alexins."  Hankin,  in  his  paper  upon  the  origin  of  these  "defen- 
sive proteids"  in  the  animal  body  (1892),  arrives  at  the  conclusion 


SUSCEPTIBILITY    AND   IMMUNITY.  237 

that  while  they  are  present  in  the  cell-free  serum  they  are  the  prod- 
uct of  certain  leucocytes — Ehrlich's  eosinophile  cells.  He  believes 
that  the  eosinophile  granules  become  dissolved  in  the  serum  and  con- 
stitute the  germicidal  proteid  which  is  shown  to  be  present  by  ex- 
periments upon  bacteria.  According  to  Hankin  the  separation  of 
these  granules  can  be  witnessed  under  the  microscope.  They  first 
accumulate  upon  one  side  of  the  cell  and  then  gradually  disappear, 
and  as  this  occurs  a  considerable  increase  in  the  bactericidal  power 
of  the  serum  can  be  demonstrated.  The  germicidal  power  of  the 
blood  serum  is  also  said  to  be  increased  when  the  number  of  leuco- 
cytes is  considerably  augmented,  as  occurs  when  a  sterilized  culture 
of  Vibrio  Metschnikovi  is  injected  subcutaneously.  Also  by  treat- 
ment which  favors  a  separation  of  the  alexin  from  the  leucocytes, 
i.e.,  a  solution  of  the  eosinophile  granules.  This  may  be  accom- 
plished by  the  injection  of  an  extract  of  the  thymus  gland  of  the 
calf ,  or  by  simpty  allowing  the  drawn  blood  to  stand  for  several  hours 
at  a  temperature  of  38°  to  40°  C. 

Buchner's  latest  communication  upon  the  subject  shows  that  he 
also  attributes  the  origin  of  the  germicidal  proteid  in  fresh  blood 
serum  to  the  leucocytes.  In  his  paper  on  "Immunity,"  read  at  the 
Eighth  International  Congress  on  Hygiene  and  Demography  (Buda- 
pest, 1894),  he  calls  attention  in  the  first  place  to  the  fact  that  a 
clearly  marked  distinction  must  be  made  between  natural  immunity 
and  acquired  immunity,  inasmuch  as  the  "  alexins  "  and  "  antitoxins  " 
have  very  different  properties.  The  first-mentioned  proteids  are  de- 
stroyed by  a  comparatively  low  temperature  (55°  to  CO0  C.),  while  the 
antitoxins  resist  a  considerably  higher  temperature,  and,  unlike  the 
alexins,  have  no  bactericidal  or  globulicidal  action.  A  very  remark- 
able fact  developed  in  Buchner's  experiments  is  that  the  blood  serum 
from  the  dog  and  from  the  rabbit,  when  mixed,  neutralize  each  other 
so  far  as  their  germicidal  power  is  concerned. 

By  injecting  sterilized  emulsions  of  wheat-flour  paste  in  the 
pleural  cavity  of  rabbits  and  dogs  Buchiier  succeeded  in  obtaining  an 
exudate  which  had  more  decided  germicidal  power  than  the  blood 
or  serum  of  the  same  animal.  This  was  evidently  due  to  the  large 
number  of  leucocytes  present,  but  not  to  their  phagocytic  action,  as 
was  shown  by  experiment.  By  freezing  the  exudate  the  leucocytes 
were  killed,  but  the  germicidal  action  of  the  fluid  was  rather  in- 
creased than  diminished  by  freezing.  While  freezing  had  no  effect 
upon  the  germicidal  action  of  the  pleural  exudate,  this  was  always 
neutralized  by  exposure  to  a  temperature  of  55°  C. 

Emmerich,  Tsuboi,  Steinmetz,  and  Low  (1892),  as  a  result  of  ex- 
tended experiments,  arrived  at  the  conclusion  that  the  germicidal 
action  of  blood  serum  "  depends  upon  a  specific  property  of  the  alkali 


238  slSCEPTIBILITY   AND   IMMUNITY. 

serumalbuiiiin,  and  that  it  is  a  purely  chemical  process."  They 
state  that  when  the  germicidal  power  is  neutralized  by  heat  it  may 
be  restored  by  the  addition  of  an  alkali.  Buclmer  repeated  the  ex- 
periments of  Emmerich  and  his  associates  and  obtained  similar  re- 
sults, but  interprets  them  differently.  According  to  him  the  serum 
does  not  regain  its  germicidal  power,  but  after  the  addition  of  an 
alkali  and  subsequent  dialyzing  the  nutritive  value  of  the  serum  is  so 
diminished  that  the  bacteria  do  not  develop  in  it. 

Pane  (1892)  has  made  experiments  which  give  additional  weight 
to  the  assumption  that  the  alkalinity  of  the  blood  is  an  important 
factor  in  accounting  for  immunity.  He  states  that  carbonate  of 
soda,  dissolved  in  water,  in  the  proportion  of  1:3,000,  has  a  de- 
cided germicidal  action  upon  the  anthrax  bacillus,  equal  to  that  of 
the  blood  serum  of  the  rabbit.  And  that  when  rabbit  serum  is  com- 
pletely neutralized  it  no  longer  has  any  injurious  action  on  anthrax 
bacilli. 

Zagari  and  Innocente  (1892)  also  arrived  at  the  conclusion  that 
the  diminished  resistance  to  anthrax  infection  resulting  from  curare 
poisoning  in  frogs,  and  from  chloral  or  alcohol  in  dogs  (Platania) ,  in 
fowls  as  a  result  of  starvation  (Canalis  and  Morpurgo),  in  white 
mice  as  a  result  of  fatigue  (Charm  and  Roger),  is,  in  fact,  due  to 
diminished  alkalinity  of  the  blood,  which  they  found  to  correspond 
with  the  increased  susceptibilit}'  resulting  from  the  causes  men- 
tioned . 

Buchner  (1892)  states  that  several  of  the  ammonium  salts,  and 
especially  ammonium  sulphate,  cause  an  increase  in  the  germicidal 
action  of  blood  serum,  and  also  increase  its  resistance  to  the  neutral- 
izing effects  of  heat.  The  experiments  of  Pansini  and  Calabrese 
(1M)4)  show,  on  the  contrary,  that  the  addition  of  uric  acid  to  blood 
serum  diminishes  its  bactericidal  activity,  as  does  also  the  presence 
of  glucose.  That  certain  infectious  diseases  are  especially  virulent 
in  |>ersons  suffering  from  diabetes  is  a  frequently  repeated  clinical 
observation. 

Van  Fodor  has  shown  by  experiment  that  the  injection  of  an 
alkali  into  the  circulation  of  a  rabbit  increases  its  resistance  to 
anthrax  infection  and  the  germicidal  activity  of  its  blood  serum. 
The  same  bacteriologist  has  found  that  when  a  rabbit  is  infected 
with  anthrax,  the  alkalinity  of  its  blood  is  notably  increased  during 
the  first  twenty-four  hours,  when  we  may  suppose  that  the  powers 
of  nature  .11-0  brought  to  bear  to  resist  the  invading  parasite,  and  that 
after  this  time  it  rapidly  diminishes.  Ten  hours  after  infection  (by 
subcutaneous  inoculation?)  the  alkalinity  of  the  blood  had  increased 
51.fi  per  rent.  Shortly  before  the  death  of  the  animal  a  diminution 
<>f  M.8  j>er  cent  was  noted.  This  diminution  was  observed  in  thirty- 


SUSCEPTIBILITY    AND    IMMUNITY.  239 

four  out  of  thirty-nine  animals  experimented  upon,  and  these  ani- 
mals succumbed  to  the  anthrax  infection  in  a  shorter  time  than  did 
the  other  five  in  which  there  was  no  such  diminution. 

It  seems  probable  that  the  germicidal  property  of  freshly  drawn 
blood  serum  is  not  due  to  its  alkalinity,  per  se,  but  to  the  fact  that 
the  germicidal  constituent  is  only  soluble  in  an  alkaline  fluid.  The 
researches  of  Vaughn,  McClintock,  and  Novy  indicate  that  this  ger- 
micidal constituent  is  a  nucleiu.  Dr.  Vaughn,  in  his  last  published 
paper  upon  "Nucleins  and  Nuclein  Therapy,"  says:  "Kossel,  of 
Berlin,  has  confirmed  our  statements  concerning  the  germicidal 
action  of  the  nucleins.  Dr.  McClintock  and  I  have  also  demon- 
strated that  the  germicidal  constituent  of  blood  serum  is  a  nuclei n. 
This  nuclein  is  undoubtedly  furnished  by  the  polynuclear  white 
corpuscles."  Denys  has  (1894)  reported  the  results  of  experi- 
ments made  in  his  laboratory  by  Van  der  Velde,  which  give  sup- 
port to  the  conclusion  reached  by  Vaughn.  In  these  experiments  a 
sterilized  culture  of  staphylococci  was  injected  into  the  pleural  cavity 
of  rabbits  in  order  to  obtain  an  exudate.  At  intervals  of  two  hours 
this  exudate  was  obtained  by  killing  one  of  the  animals  in  the  series 
experimented  upon,  and  at  the  same  time  blood  from  the  animal  was 
secured.  Both  the  exudate  and  the  blood  were  placed  in  a  centrifugal 
machine,  in  order  to  obtain  a  serum  free  from  corpuscular  elements. 
The  germicidal  activity  of  the  serum  was  then  tested.  The  general 
result  of  the  experiments  was  to  show  that  the  longer  the  interval 
after  the  injection  into  the  pleural  cavity  the  more  potent  the  ger- 
micidal activity  of  the  exudate  became,  and  that  there  was  no  corre- 
sponding increase  in  the  activity  of  the  blood  serum  obtained  from 
the  circulation.  At  the  end  of  ten  or  twelve  hours,  the  serum  from 
the  exudate  killed  all  of  the  staphylococci  in  a  bouillon  culture  twenty 
times  as  great  in  quantity  as  the  germicidal  serum  used  in  the  ex- 
periment. The  absence  of  any  increase  in  germicidal  power  in  the 
blood  serum  taken  from  the  general  circulation  shows  that  the  nota- 
ble increase  manifested  by  the  exudate  was  due  to  local  causes;  and 
as  a  matter  of  fact  it  corresponded  with  an  increase  in  the  number  of 
leucocytes  as  found  in  the  pleural  exudate. 

Thus  it  will  be  seen  that  the  independent  researches  of  Hankin, 
of  Buchner,  of  Vaughn,  and  of  other  competent  bacteriologists,  have 
led  them  to  the  same  ultimate  result  so  far  as  the  origin  of  the  ger- 
micidal constituent  of  the  blood  is  concerned,  and  that  the  leucocytes 
appear  to  play  an  important  role  in  the  protection  of  the  animal  body 
from  invasion  by  bacteria  (natural  immunity). 

It  has  been  shown  by  several  investigators  that  the  number  of 
leucocytes  increases  in  certain  infectious  diseases,  and  this  increase, 
together  with  an  increased  alkalinity  of  the  blood,  which  has  here- 


240  SUSCEPTIBILITY   AND   IMMUNITY. 

tofore  been  referred  to,  appears  to  be  a  provision  of  nature  for  over- 
coming the  infection  which  has  already  occurred. 

It  has  been  demonstrated  by  experiment  that  naturally  immune 
animals  may  be  infected  by  the  addition  of  certain  substances  to  cul- 
tures of  pathogenic  bacteria.  Thus  Arloing  was  able  to  induce  symp- 
tomatic anthrax  in  animals  naturally  immune  for  this  disease  by 
mixing  with  his  cultures  various  chemical  substances,  such  as  car- 
bolic acid,  pyrogallic  acid,  and  especially  lactic  acid  (twenty  per 
cent).  Leo  has  shown  that  white  mice,  which  are  not  subject  to 
the  pathogenic  action  of  the  glanders  bacillus,  may  be  rendered  sus- 
ceptible by  feeding  them  for  some  time  upon  phloridzin,  which  gives 
rise  to  an  artificial  diabetes,  and  causes  the  tissues  to  become  im- 
pregnated with  sugar. 

Bouchard  has  found  that  very  small  doses  of  a  pure  culture  of 
Bacillus  pyocyaneus  are  fatal  to  rabbits  when  at  the  same  time  a 
considerable  quantity  of  a  filtered  culture  of  the  same  bacillus  is  in- 
jected into  a  vein.  The  animal  could  have  withstood  the  filtered 
culture  alone,  or  the  bacillus  injected  beneath  its  skin ;  but  its  resist- 
ing power — natural  immunity — is  overcome  by  the  combined  action 
of  the  living  bacilli  and  the  toxic  substances  contained  in  the  filtered 
culture.  The  same  result  may  be  obtained  by  injecting  sterilized 
cultures  of  a  different  microorganism.  Thus  Roger  has  shown  that 
the  rabbit,  which  has  a  natural  immunity  against  symptomatic 
anthrax,  succumbs  to  infection  when  inoculated  with  a  culture  of  the 
bacillus  of  this  disease,  if  at  the  same  time  it  receives  an  injection  of 
a  sterilized  or  non-sterilized  culture  of  Bacillus  prodigiosus.  Monti 
has  succeeded  in  killing  animals  with  old  and  attenuated  cultures  of 
Streptococcus  pyogenes,  or  of  Staphylococcus  pyogenes  aureus,  by  in- 
jecting at  the  same  time  a  culture  of  Proteus  vulgaris.  In  a  similar 
way,  it  seems  probable,  the  normal  resistance  of  man  to  infection  by 
certain  pathogenic  bacteria  may  be  overcome.  Thus  when  water 
contaminated  by  the  presence  of  the  typhoid  bacillus  is  used  for 
drinking  by  the  residents  of  a  certain  town  or  district,  not  all  of 
those  who  in  this  way  are  exposed  to  infection  contract  typhoid 
fever;  and  among  those  who  do,  there  is  good  reason  to  believe  that, 
in  certain  cases  at  least,  the  result  depends  upon  an  additional  factor 
of  the  kind  suggested  by  the  above-mentioned  experiments—  e.g.,  the 
consumption  of  food  containing  putrefactive  products,  or  the  respi- 
r; it  inn  of  an  atmosphere  containing  volatile  products  of  putrefaction. 

The  natural  immunity  of  healthy  animals  may  also  be  neutralized 
by  other  agencies  which  have  a  depressing  effect  upon  the  vital  re- 
sisting power.  Thus  Nocard  and  Roux  found  by  experiment  that  an 
attenuated  culture  of  the  anthrax  bacillus,  which  was  not  fatal  to 
guinea-pigs,  killed  these  animals  when  injected  into  the  muscles  of 


SUSCEPTIBILITY   AND    IMMUNITY.  241 

the  thigh  after  they  had  been  bruised  by  mechanical  violence. 
Abarrin  and  Roger  found  that  white  rats,  which  are  not  susceptible 
to  anthrax,  became  infected  and  frequently  died  if  they  were  ex- 
hausted, previous  to  inoculation,  by  being  compelled  to  turn  a  revolv- 
ing wheel  for  a  considerable  time.  Pasteur  found  that  fowls,  which 
have  a  natural  immunity  against  anthrax,  become  infected  and 
perish  if  they  are  subjected  to  artificial  refrigeration  after  inocula- 
tion. This  has  been  confirmed  by  the  more  recent  experiments  of 
Wagner  (1891).  According  to  Canalis  and  Morpurgo,  pigeons 
which  are  enfeebled  by  inanition  eaily  contract  anthrax  as  a  result 
of  inoculation.  Arloing  states  that  sheep  which  have  been  freely 
bled  contract  anthrax  more  easily  than  others ;  and  Serafini  found 
that  when  dogs  were  freely  bled  the  bacillus  of  Friedlander,  injected 
into  the  trachea  or  the  pleural  cavity,  entered,  and  apparently  mul- 
tiplied to  some  extent  in  the  blood,  whereas  without  such  previous 
bleeding  they  were  not  to  be  found  in  the  circulating  fluid.  Certain 
anesthetic  agents  have  been  shown  also  to  produce  a  similar  result. 
Platania  communicated  anthrax  to  immune  animals — dogs,  frogs, 
pigeons — by  bringing  them  under  the  influence  of  curare,  chloral,  or 
alcohol;  and  Wagner  obtained  similar  results  in  his  experiments 
upon  pigeons  to  which  he  had  administered  chloral.  In  man,  clini- 
cal experience  shows  that  those  who  are  addicted  to  the  excessive  use 
of  alcohol  are  especially  liable  to  contract  certain  infectious  diseases 
—pneumonia,  erysipelas,  yellow  fever,  etc. 

The  micrococcus  of  pneumonia  is  habitually  present  in  the  sali- 
vary secretions  of  many  healthy  individuals,  and  it  is  evident  that 
an  attack  of  pneumonia  does  not  depend  alone  upon  the  presence  of 
this  micrococcus,  which  has,  nevertheless,  been  conclusively  shown 
to  be  the  usual  infectious  agent  in  cases  of  croupous  pneumonia.  No 
doubt  the  introduction  of  the  pathogenic  micrococcus  to  the  vulner- 
able point — the  lungs — is  an  essential  factor  in  the  development  of  a 
case  of  pneumonia,  but  there  is  reason  to  believe  that  there  are  other 
factors  equally  essential.  Thus  it  is  well  known  that  an  attack  of 
pneumonia  often  results  from  exposure  to  cold,  which  may  act  as  an 
exciting  cause ;  and,  also,  that  a  recent  attack  of  an  acute  febrile 
disease — especially  measles — constitutes  a  predisposing  cause.  It  is 
generally  recognized  that  malnutrition,  want  of  exercise,  insanitary 
surroundings,  and  continued  respiration  of  an  atmosphere  loaded 
with  dust,  as  in  cotton  mills,  or  a  recent  attack  of  pneumonia,  con- 
stitute predisposing  causes  to  tubercular  infection  by  way  of  the 
lungs. 

While  natural  immunity  may  be  overcome  by  the  various  depress- 
ing agencies  referred  to,  it  is  also  true  that  it  has  only  a  relative 
value  in  the  absence  of  these  predisposing  causes,  and  may  be  over- 
16 


242  SUSCEPTIBILITY   AND   IMMUNITY. 

come  by  unusual  virulence  of  the  pathogenic  infectious  agent,  or  by 
the  introduction  into  the  body  of  an  excessive  amount  of  a  pure  cul- 
ture of  the  same. 

The  pathogenic  potency  of  known  disease  germs  varies  as  widely 
as  does  the  susceptibility  of  individuals  to  their  specific  action.  In 
general  it  may  be  said  that  the  more  recently  the  germ  comes  from 
a  developed  case  of  the  disease  to  which  it  gives  rise  the  more  viru- 
lent it  is,  and  the  longer  it  has  been  cultivated  outside  of  the  animal 
body  the  more  attenuated  is  its  pathogenic  power.  Thus  when  the 
discharges  of  a  typhoid  fever  patient  find  their  way  directly  to  a 
water-supply  of  limited  amount  a  large  proportion  of  those  who 
drink  the  water  are  likely  to  be  attacked;  but  when  a  considerable 
interval  of  time  has  elapsed  since  the  contamination  occurred, 
although  the  germs  may  still  be  present,  the  liability  to  attack  is 
much  less  on  account  of  diminished  pathogenic  virulence. 

The  development  of  an  attack  also  depends,  to  some  extent,  upon 
the  number  of  germs  introduced  into  a  susceptible  individual  at  one 
time.  The  resources  of  nature  may  be  sufficient  to  dispose  of.  a  few 
bacilli,  while  a  large  number  may  overwhelm  the  resisting  power  of 
the  individual. 

The  experiments  of  Cheyne  (1886)  show  that  in  the  case  of  very 
pathogenic  species  a  single  bacillus,  or  at  least  a  very  small  number, 
introduced  beneath  the  skin,  may  produce  fatal  infection  in  a  very 
susceptible  animal,  while  greater  numbers  are  required  in  those  less 
susceptible.  Thus  a  guinea-pig  succumbed  to  general  infection  after 
being  inoculated  subcutaneously  with  anthrax  blood  diluted  to  such 
an  extent  that,  by  estimation,  only  one  bacillus  was  present  in  the 
fluid  injected ;  and  a  similar  result  was  obtained  in  mice  with  Bacillus 
murisepticus.  In  the  case  of  the  microbe  of  fowl  cholera  (Bacillus 
septica3mia  hemorrhagicaB) ,  Cheyne  found  that  for  rabbits  the  fatal 
dose  was  300,000  or  more,  that  from  100,000  to  30,000  cause  a  local 
abscess,  and  that  less  than  10,000  produce  no  appreciable  effect.  The 
common  saprophyte,  Proteus  vulgaris,  was  found  to  be  pathogenic 
for  rabbits  when  injected  into  the  dorsal  muscles  in  sufficient  num- 
bers. But,  according  to  the  estimates  made,  225,000,000  were  re- 
quired to  cause  death,  while  doses  of  from  9,000,000  to  112,000,000 
produced  a  local  abscess,  and  less  than  9,000,000  gave  an  entirely 
negative  result. 

ACQUIRED   IMMUNITY. 

It  has  long  been  known  that,  in  a  considerable  number  of  infec- 
tious diseases,  a  single  attack,  however  mild,  affords  protection 
against  subsequent  attacks  of  the  same  disease;  that  in  some  cases 
this  protection  appears  to  be  permanent,  lasting  during  the  life  of  the 


SUSCEPTIBILITY    AND   IMMUNITY.  213 

individual;  that  in  others  it  is  more  or  less  temporary,  as  shown  by 
the  occurrence  of  a  subsequent  attack. 

The  protection  afforded  by  a  single  attack  not  only  differs  in  dif- 
ferent diseases,  but  in  the  same  disease  varies  greatly  in  different 
individuals.  Thus  certain  individuals  have  been  known  to  suffer 
several  attacks  of  small-pox  or  of  scarlet  fever,  although,  as  a  rule,  a 
single  attack  is  protective.  Exceptional  susceptibility  or  insuscepti- 
bility may  be  not  only  an  individual  but  a  family  characteristic,  or 
it  may  belong  to  a  particular  race. 

In  those  diseases  in  which  second  attacks  are  not  infrequent,  as, 
for  example,  in  pneumonia,  in  influenza,  or  in  Asiatic  cholera,  it  is 
difficult  to  judge  from  clinical  experience  whether  a  first  attack  exerts 
any  protective  influence.  But  from  experiments  upon  the  lower  ani- 
mals we  are  led  to  believe  that  a  certain  degree  of  immunity,  lasting 
for  a  longer  or  shorter  time,  is  afforded  by  an  attack  of  pneumonia 
or  of  cholera,  and  probably  of  all  infectious  diseases  due  to  bacterial 
parasites.  In  the  malarial  fevers,  which  are  due  to  a  parasite  of  a 
different  class,  one  attack  affords  no  protection,  but  rather  predis- 
poses to  a  subsequent  attack. 

In  those  diseases  in  which  a  single  attack  is  generally  recognized 
as  being  protective,  exceptional  cases  occur  in  which  subsequent 
attacks  are  developed  as  a  result  of  unusual  susceptibility  or  expo- 
sure under  circumstances  especially  favorable  to  infection.  Maiselis 
(1894)  has  gone  through  the  literature  accessible  to  him  for  the 
purpose  of  determining  the  frequency  with  which  second  attacks 
occur  in  the  various  diseases  below  mentioned.  The  result  is  as 
follows : 

Second  Third         Fourth  „,  .   , 

Attacks.         Attacks.      Attacks. 

Small-pox 505  9  0  514 

Scarlet  fever 29  4  0  33 

Measles 36  1  0  37 

Typhoid  fever 202  5  1  208 

Cholera 29  3  2  34 

These  figures  support  the  view  generally  entertained  by  physi- 
cians that  second  attacks  of  scarlet  fever  and  of  measles  are  compar- 
atively rare,  while  second  attacks  of  small-pox  are  not  infrequently 
observed.  Considering  the  very  large  number  of  cases  of  typhoid 
fever  which  occur  annually  in  all  parts  of  Europe  and  America,  the 
number  of  second  attacks  collected  does  not  bear  a  very  large  propor- 
tion to  the  total  number  taken  sick,  although  the  recorded  cases,  of 
course,  fall  far  short  of  the  total  number  of  second  attacks  of  this 
and  the  other  diseases  mentioned. 

The  second  attacks  of  cholera  recorded  are  not  numerous,  and,  no 
doubt,  a  carefullly  conducted  investigation  made  in  the  areas  of  en- 


244  SUSCEPTIBILITY   AND   IMMUNITY. 

demic  prevalence  of  this  disease  would  show  that  second  attacks  are 
more  common  than  is  indicated  by  these  figures. 

That  immunity  may  result  from  a  comparatively  mild  attack  as 
well  as  from  a  severe  one  is  a  matter  of  common  observation  in  the 
case  of  small-pox,  scarlet  fever,  yellow  fever,  etc. ;  and  since  the  dis- 
covery of  Jenner  we  have  in  vaccination  a  simple  method  of  produc- 
ing immunity  in  the  first-mentioned  disease.  The  acquired  immunity 
resulting  from  vaccination  is  not,  however,  as  complete  or  as  per- 
manent as  that  which  results  from  an  attack  of  the  disease. 

These  general  facts  relating  to  acquired  immunity  from  infectious 
diseases  constituted  the  principal  portion  of  our  knowledge  with  re- 
ference to  this  important  matter  up  to  the  time  that  Pasteur  (1880) 
demonstrated  that  in  the  disease  of  fowls  known  as  chicken  cholera, 
which  he  had  proved  to  be  due  to  a  specific  microorganism,  a  mild 
attack  followed  by  immunity  may  be  induced  by  inoculation  with  an 
"  attenuated  virus" — i.e.,  by  inoculation  with  a  culture  of  the  patho- 
genic microorganism  the  virulence  of  which  had  been  so  modified 
that  it  gave  rise  to  a  comparatively  mild  attack  of  the  disease  in 
question.  Pasteur's  original  method  of  obtaining  an  attenuated  virus 
consisted  in  exposing  his  cultures  for  a  considerable  time  to  the  ac- 
tion of  atmospheric  oxygen.  It  has  since  been  ascertained  that  the 
same  result  is  obtained  with  greater  certainty  by  exposing  cultures 
for  a  given  time  to  a  temperature  slightly  below  that  which  would 
destroy  the  vitality  of  the  pathogenic  microorganism,  and  also  by  ex- 
posure to  the  action  of  certain  chemical  agents  (see  Part  Second,  p. 
124). 

Pasteur  at  once  comprehended  the  importance  of  his  discovery, 
and  inferred  that  what  was  true,  of  one  infectious  germ  disease  was 
likely  to  be  true  of  others.  Subsequent  researches,  by  this  savant 
and  by  other  bacteriologists,  have  justified  this  anticipation,  and  the 
demonstration  has  already  been  made  for  a  considerable  number  of 
similar  diseases — anthrax,  symptomatic  anthrax,  rouget. 

A  virus  which  has  been  attenuated  artificially — by  heat,  for  ex- 
ample— may  be  cultivated  through  successive  generations  without  re- 
gaining its  original  virulence.  As  this  virulence  depends,  to  a  con- 
siderable extent  at  least,  upon  the  formation  of  toxic  products  during 
the  development  of  the  pathogenic  microorganism,  we  naturally  infer 
that  diminished  virulence  is  due  to  a  diminished  production  of  these 
toxic  substances. 

There  is  reason  to  believe  that  a  natural  attenuation  of  virulence 
may  occur  in  pathogenic  bacteria  which  are  able  to  lead  a  sapro- 
phytic  existence  during  their  multiplication  external  to  the  bodies  of 
living  animals,  and  the  comparatively  mild  character  of  some  epi- 
demics is  probably  due  to  this  fact. 


SUSCEPTIBILITY   AND   IMMUNITY.  245 

Again,  cultivation  within  the  body  of  a  living  animal  may,  in 
certain  cases,  cause  a  diminution  in  the  virulence  of  a  pathogenic 
microorganism.  Thus  Pasteur  and  Thuiller  have  shown  that  the 
microbe  of  rouget  when  inoculated  into  a  rabbit  kills  the  animal,  but 
that  its  pathogenic  virulence  is  nevertheless  so  modified  that  a  cul- 
ture made  from  the  blood  of  a  rabbit  killed  by  it  is  a  suitable  "  vac- 
cine "  for  the  pig. 

On  the  other  hand,  we  have  experimental  evidence  that  the  viru- 
lence of  attenuated  cultures  may  be  reestablished  by  passing  them 
through  the  bodies  of  susceptible  animals.  Thus  a  culture  of  the 
bacillus  of  rouget,  attenuated  by  having  been  passed  through  the 
body  of  a  rabbit,  is  restored  to  its  original  virulence  by  passing  it 
through  the  bodies  of  pigeons.  And  a  culture  of  the  anthrax  bacillus 
which  will  not  kill  an  adult  guinea-pig  may  be  fatal  to  a  very  young 
animal  of  the  same  species  or  to  a  mouse,  and  the  bacillus  cultivated 
from  the  blood  of  such  an  animal  will  be  found  to  have  greatly  in- 
creased virulence. 

In  Pasteur's  inoculations  against  anthrax  "attenuated"  cultures 
are  employed  which  contain  the  living  pathogenic  germ  as  well  as 
the  toxic  products  developed  during  its  growth.  Usually  two  inocu- 
lations are  made  with  cultures  of  different  degrees  of  attenuation — 
that  is  to  say,  with  cultures  in  which  the  toxic  products  are  formed 
in  less  amount  than  in  virus  of  full  power.  The  most  attenuated 
virus  is  first  injected,  and  after  some  time  the  second  vaccine,  which 
if  injected  first  might  have  caused  a  considerable  mortality.  The 
animal  is  thus  protected  from  the  pathogenic  action  of  the  most 
virulent  cultures. 

Now,  it  has  been  shown  by  recent  experiments  that  a  similar  im- 
munity may  result  from  the  injection  into  a  susceptible  animal  of  the 
toxic  products  contained  in  a  virulent  culture,  independently  of  the 
living  bacteria  to  which  they  owe  their  origin.  Chauveau,  in  1880, 
ascertained  that  if  pregnant  ewes  are  protected  against  anthrax  by 
inoculation  with  an  attenuated  virus,  their  lambs,  when  born,  also 
give  evidence  of  having  acquired  an  immunity  from  the  disease.  As 
the  investigations  of  Davaine  seemed  to  show  that  the  anthrax 
bacillus  cannot  pass  through  the  placenta  from  the  mother  to  the 
foetus,  the  inference  seemed  justified  that  the  acquired  immunity  of 
the  latter  was  due  to  some  soluble  substance  which  could  pass  the 
placental  barrier.  More  recent  researches  by  Strauss  and  Chamber- 
lain, Malvoz  and  Jacquet,  and  others,  show  that  the  placenta  is  not 
such  an  impassable  barrier  for  bacteria  as  was  generally  believed  at 
the  time  of  Chauveau's  experiments,  so  that  these  cannot  be  accepted 
as  establishing  the  inference  referred  to.  But,  as  stated,  we  have 
more  recent  experimental  evidence  which  shows  that  immunity  may 


246  SUSCEPTIBILITY  AND   IMMUNITY. 

result  from  the  introduction  into  the  bodies  of  susceptible  animals  of 
the  toxic  substances  produced  by  certain  pathogenic  bacteria.  The 
first  satisfactory  experimental  evidence  of  this  important  fact  was 
obtained  by  Salmon  and  Smith  in  1886,  who  succeeded  in  making 
pigeons  immune  from  the  pathogenic  effects  of  cultures  of  the  bacil- 
lus of  hog  cholera  by  inoculating  them  with  sterilized  cultures  of 
this  bacillus.  In  1888  Roux  reported  similar  results  obtained  by  in- 
jecting into  susceptible  animals  sterilized  cultures  of  the  anthrax 
bacillus.  Behring  and  Kitasato,  in  1890,  reported  their  success  in 
establishing  immunity  against  virulent  cultures  of  the  bacillus  of 
tetanus  and  the  diphtheria  bacillus  by  inoculating  susceptible  ani- 
mals with  filtered,  germ-free  cultures  of  these  pathogenic  bacteria. 

In  1892  Behring,  Kitasato,  and  Wassermann  published  the  re- 
sults of  interesting  experiments  with  a  bouillon  made  from  the 
thymus  gland  of  the  calf.  They  found  that  the  tetanus  bacillus  cul- 
tivated in  this  bouillon  did  not  form  spores  and  had  comparatively 
little  virulence.  Mice  or  rabbits  inoculated  with  it  in  small  doses — 
0.001  to  0.2  cubic  centimetre  for  a  mouse — proved  to  be  subsequently 
immune.  And  the  blood  serum  of  an  immune  rabbit  injected  into 
the  peritoneal  cavity  of  a  mouse — 0.1  to  0.5  cubic  centimetre — was 
found  to  give  it  immunity  from  the  pathogenic  action  of  a  virulent 
culture  of  the  tetanus  bacillus.  Similar  results  were  obtained  with 
several  other  pathogenic  bacteria  cultivated  in  the  thymus  bouillon- 
spirillum  of  cholera,  bacillus  of  diphtheria,  typhoid  bacillus.  We 
give  here  the  directions  for  preparing  the  thymus  bouillon  as  used  by 
the  authors  named : 


Two  or  three  thymus  glands  are  chopped  into  small  pieces  immediately 
after  they  are  taken  from  the  animal.  An  equal  part  of  distilled  water  is 
added  to  the  mass  and  stirred  for  some  time  ;  it  is  then  placed  in  an  ice  chest 
for  twelve  hours.  The  juices  are  now  expressed  through  gauze  by  means  of 
a  flesh  press.  A  clouded,  slimy  fluid  is  obtained,  which  constitutes  a  stock 
solution.  This  is  diluted  with  water,  and  a  certain  quantity  of  carbonate  of 
soda  is  added  to  the  solution  before  sterilization.  By  this  means  coagulation 
and  precipitation  of  the  active  substance  from  the  thymus  gland  are  avoided. 
The  exact  amount  of  water  and  of  sodium  carbonate  required  to  prevent  pre- 
cipitation must  be  determined  by  experiment,  as  it  differs  for  different  glands. 
Usuall v  an  eoual  portion  of  water  and  sufficient  soda  solution  to  turn  litmus 
paper  feeblv  blue  will  give  the  desired  result.  The  liquid  is  now  heated  in 
a  large  flask,  which  is  left  for  fifteen  minutes  in  the  steam  sterilizer.  The 
liquid  is  allowed  to  cool  and  then  filtered  through  fine  linen  to  remove  any 
suspended  coagula  ;  the  filtrate  has  a  milky  opalescence.  It  is  now  placed 
in  test  tubes  and  again  sterilized.  The  active  principle  is  precipitated  by  the 
addition  of  a  few  drops  of  acetic  acid. 

In  Pasteur's  inoculations  against  hydrophobia,  made  subsequently 
to  infection  by  the  bite  of  a  rabid  animal,  an  attenuated  virus  is  in- 


SUSCEPTIBILITY   AND   IMMUNITY.  247 

troduced  subcutaneously  in  considerable  quantity  by  daily  injections, 
and  immunity  is  established  during  the  interval — so-called  period  of 
incubation— which  usually  occurs  between  the  date  of  infection  and 
the  development  of  the  disease.  That  the  immunity  in  this  case  also 
depends  upon  the  introduction  of  a  chemical  substance  present  in  the 
desiccated  spinal  cord  of  rabbits  which  have  succumbed  to  rabies, 
which  is  used  in  these  inoculations,  is  extremely  probable.  But,  as 
the  germ  of  rabies  has  not  been  isolated  or  cultivated  artificially,  this 
has  not  yet  been  demonstrated.  Wooldridge  claims  to  have  made 
susceptible  animals  immune  against  anthrax  by  inoculating  them 
with  an  aqueous  extract  of  the  testicle  or  of  the  thymus  gland  of 
healthy  animals. 

We  may  mention  also  the  interesting  results  obtained  by  Em- 
merich, Freudenreich,  and  others,  who  have  shown  that  an  anthrax 
infection  in  a  susceptible  animal  inoculated  with  a  virulent  culture 
may  be  made  to  take  a  modified  and  non-fatal  course  by  the  simul- 
taneous or  subsequent  inoculation  of  certain  other  non-pathogenic 
bacteria — streptococcus  of  erysipelas,  Bacillus  pyocyaneus. 

In  a  series  of  experiments  made  by  the  writer  some  years  ago 
evidence  was  obtained  that,  under  certain  circumstances,  immunity 
from  the  effects  of  one  pathogenic  bacillus  may  be  obtained  by  the 
previous  injection  of  a  pure  culture  of  a  different  species.  In  the 
experiments  referred  to  injections  into  the  cavity  of  the  abdomen  of 
a  culture  of  Bacillus  pyocyaneus  protected  rabbits  from  the  lethal 
effects  of  Bacillus  cuniculicida  Havaniensis,  when  subsequently  in- 
jected into  the  cavity  of  the  abdomen  in  such  amount  (one  cubic 
centimetre  of  a  bouillon  culture)  as  invariably  proved  fatal  in  rabbits 
not  protected  by  such  injections. 

Before  considering  the  theories  which  have  been  offered  in  expla- 
nation of  acquired  immunity  it  is  desirable  to  call  attention  to  certain 
observations  which  have  been  made  during  the  past  few  years  relat- 
ing to  "chemiotaxis." 

The  term  chemiotaxis  was  first  used  by  Pfeffer  to  designate  the 
property,  observed  by  himself  and  others,  which  certain  living  cells 
exhibit  with  reference  to  non-living  organic  material,  and  by  virtue 
of  which  they  approach  or  recede  from  certain  substances.  The 
chemiotaxis  is  said  to  be  positive  when  the  living  cell  approaches,  and 
negative  when  it  recedes  from,  a  chemical  substance.  As  examples 
of  this  we  may  mention  the  approach  of  motile  bacteria  to  nutrient 
material  or  to  the  surface  of  a  liquid  medium  where  they  find  the 
oxygen  required  for  their  vital  activities  ;  and  of  leucocytes  to  cer- 
tain substances  when  these  are  introduced  beneath  the  skin  of  warm- 
er cold-blooded  animals.  This  subject  has  recently  received  much 


248  SUSCEPTIBILITY   AND   IMMUNITY. 

attention  and  has  been  studied  especially  by  Ali-Cohen,  Massart  and 
Bordet,  Gabritchevski,  and  others. 

According  to  Gabritchevski,  the  following  substances  have  a  neg- 
ative chemiotaxis  for  the  leucocytes  :  Sodium  chloride  in  ten-per-cent 
solution,  alcohol  in  ten-per-cent  solution,  quinine,  lactic  acid,  gly- 
cerin, chloroform,  bile.  On  the  other  hand,  a  positive  chemiotaxis 
is  excited  by  sterilized  or  non-sterilized  cultures  of  various  bacteria. 
This  is  shown  by  the  fact  that  when  a  small  capillary  tube,  closed  at 
one  end,  which  contains  the  substance  to  be  tested,  is  introduced  be- 
neath the  skin  of  an  animal,  the  leucocytes  are  repelled  from  the  tube 
by  certain  substances,  while  those  which  incite  positive  chemiotaxis 
cause  them  to  enter  the  tube  in  great  numbers.  The  experiments  of 
Buchner  seem  to  show  that  the  positive  chemiotaxis  induced  by 
sterilized  cultures  of  bacteria  introduced  beneath  the  skin  of  an 
animal,  is  due  to  the  proteid  contents  of  the  cells  rather  than  to  the 
chemical  products  elaborated  as  a  result  of  their  vital  activity.  But 
that  such  chemical  products  may,  in  some  instances  at  least,  produce 
a  positive  chemiotaxis  independently  of  the  bacteria  is  shown  by 
the  experiments  of  Gabritchevski  with  filtered  cultures  of  Bacillus 
pyocyaneuc — confirmed  by  Massart  and  Bordet. 

An  important  observation  made  by  Bouchard,  and  confirmed  by 
Massart  and  Bordet,  is  the  following :  When  a  tube  containing  a  cul- 
ture of  Bacillus  pyocyaneus  is  introduced  beneath  the  skin  of  a  rabbit 
it  is  found,  at  the  end  of  a  few  hours,  to  contain  a  great  number  of 
leucocytes.  But  if  immediately  after  its  introduction  ten  cubic  centi- 
metres of  a  sterilized  culture  of  the  same  bacillus  are  injected  into  the 
circulation  through  a  vein,  very  few  leucocytes  enter  the  tube  intro- 
duced beneath  the  skin — that  is,  the  chemiotaxis  of  the  leucocytes 
for  the  bacilli  contained  in  the  tube  has  been  neutralized  by  injecting 
a  considerable  quantity  of  the  soluble  products  of  the  same  bacillus 
into  the  circulation. 

Buchner,  having  shown  that  the  bacterial  cells  contain  a  proteid 
substance  which  attracts  the  leucocytes,  experimented  with  various 
other  proteids  and  found  that  gluten,  casein  from  wheat,  and  legumin 
from  peas  had  a  similar  effect.  Starch  has  no  effect,  but  a  mass  of 
flour,  made  from  wheat  or  from  peas,  introduced  beneath  the  skin  of 
a  rabbit  or  of  a  guinea-pig,  with  antiseptic  precautions,  in  the  course 
nf  ;i  day  or  t\v<>  is  enveloped  and  penetrated  by  immense  numbers  of 
leucocytes.  If,  instead  of  introducing  these  substances  which  induce 
positive  chemiotaxis  beneath  the  skin,  they  are  injected  into  the  cir- 
culation, Buchner  has  shown  that  a  great  increase  in  the  number  of 
leucocytes  occurs. 


SUSCEPTIBILITY   AND    IMMUNITY.  249 

THEORIES   OF   IMMUNITY. 

Exhaustion  Theory. — For  a  time  Pasteur  supported  the  view 
that  during  an  attack  of  an  infectious  disease  the  pathogenic  micro- 
organism, in  its  multiplication  in  the  body  of  a  susceptible  animal, 
exhausts  the  supply  of  some  substance  necessary  for  its  development, 
that  this  substance  is  not  subsequently  reproduced,  and  that  conse- 
quently the  same  pathogenic  germ  cannot  again  multiply  in  the  body 
of  the  protected  animal.  This  view  is  sustained  in  a  memoir  pub- 
lished in  the  Comptes  Rendus  of  the  French  Academy  in  1880,  in 
which  Pasteur  says  : 

"  It  is  the  life  of  a  parasite  in  the  interior  of  the  body  which  produces  the 
malady  commonly  called  ' cholera  des  ponies,'  and  which  causes  death. 
From  the  moment  when  this  culture  (i.e.,  the  multiplication  of  the  parasite) 
is  no  longer  possible  in  the  fowl  the  sickness  cannot  appear.  The  fowls  are 
then  in  the  constitutional  state  of  fowls  not  subject  to  be  attacked  by  the 
disease.  These  last  are  as  if  vaccinated  from  birth  for  this  malady,  because 
the  foetal  evolution  has  riot  introduced  into  their  bodies  the  material  neces- 
sary to  support  the  life  of  the  microbe,  or  these  nutritive  materials  have 
disappeared  at  an  early  age. 

"Certainly  one  should  not  be  surprised  that  there  may  be  constitutions 
sometimes  susceptible  and  sometimes  rebellious  to  inoculation — that  is  to 
say,  to  the  cultivation  of  a  certain  virus — when,  as  I  have  announced  in  my 
first  note,  one  sees  a  preparation  of  beer  yeast  made,  exactly  like  one  from 
the  muscles  of  fowls  (bouillon),  to  show  itself  absolutely  unsuited  for  the  cul- 
tivation of  the  parasite  of  fowl  cholera,  while  it  is  admirably  adapted  to  the 
cultivation  of  a  multitude  of  microscopic  species,  notably  to  the  bacteride 
charbonneuse  (Bacillus  anthracis). 

"The  explanation  to  which  these  facts  conduct  us,  as  well  of  the  consti- 
tutional resistance  of  some  individuals  as  of  the  immunity  produced  by 
protective  inoculations,  is  only  natural  when  we  consider  that  every  culture, 
in  general,  modifies  the  medium  in  which  it  is  effected— a  modification  of 
the  soil  when  it  relates  to  ordinary  plants;  a  modification  of  plants  and  ani- 
mals when  it  relates  to  their  parasites  ;  a  modification  of  our  culture  liquids 
when  it  relates  to  muce'dines,  vibrioniens,  or  ferments. 

' '  These  modifications  are  manifested  and  characterized  by  the  circum- 
stance that  new  cultivations  of  the  same  species  in  these  media  become 
promptly  difficult  or  impossible.  If  we  sow  chicken  bouillon  with  the  mi- 
crobe of  fowl  cholera,  and,  after  three  or  four  days,  filter  the  liquid  in  order 
to  remove  all  trace  of  the  microbe,  and  subsequently  sow  anew  in  the  fil- 
tered liquid  this  parasite,  it  will  be  found  quite  powerless  to  resume  the  most 
feeble  development.  The  liquid,  which  is  perfectly  limpid  after  being  fil- 
tered, retains  its  limpidity  indefinitely. 

"How  can  we  fail  to  believe  that  by  cultivation  in  the  fowl  of  the  atten- 
uated virus  we  place  its  body  in  the  state  of  this  filtered  liquid  which  can 
no  longer  cultivate  the  microbe  ?  The  comparison  can  be  pushed  still 
further;  for  if  we  filter  the  bouillon  containing  the  microbe  in  full  develop- 
ment, not  on  the  fourth  day  of  culture,  but  on  the  second,  the  filtered  liquid 
will  still  be  able  to  support  the  development  of  the  microbe,  although  with 
less  energy  than  at  the  outset.  We  comprehend,  then,  that  after  a  cultiva- 
tion of  the  modified  (attenue)  microbe  in  the  body  of  the  fowl  we  may  not 
have  removed  from  all  parts  of  its  body  the  aliment  of  the  microbe.  That 
which  remains  will  permit,  then,  a  new  culture,  but  in  a  more  restricted 
measure. 

"This  is  the  effect  of  a  first  inoculation  ;  subsequent  inoculations  will 


250  SUSCEPTIBILITY   AND   IMMUNITY. 

remove  progressively  all  the  material  necessary  for  the  development  of  the 
parasite. " 

In  discussing  this  theory,  in  a  paper  published  in  the  American 
Journal  of  the  Medical  Sciences  (April,  1881),  the  writer  says: 

"Let  us  see  where  this  hypothesis  leads  us.  In  the  first  place,  we  must 
have  a  material  of  small-pox,  and  a  material  of  measles,  and  a  material  of 
scarlet  fever,  etc.,  etc.  Then  we  must  admit  that  each  of  these  different 
materials  has  been  formed  in  the  system  and  stored  up  for  these  emergencies 
— attacks  of  the  diseases  in  question — for  we  can  scarcely  conceive  that  they 
were  all  packed  away  in  the  germ  cell  of  the  mother  and  the  sperm  cell  of 
the  father  of  each  susceptible  individual.  If,  then,  these  peculiar  materials 
have  been  formed  and  stored  up  during  the  development  of  the  individual, 
how  are  we  to  account  for  the  fact  that  no  new  production  takes  place  after 
an  attack  of  any  one  of  the  diseases  in  question  ? 

"  Again,  how  shall  we  account  for  the  fact  that  the  amount  of  material 
which  would  nourish  the  small-pox  germ,  to  the  extent  of  producing  a  case 
of  confluent  small-pox,  may  be  exhausted  by  the  action  of  the  attenuated 
virus  (germ)  introduced  by  vaccination  ?  Pasteur's  comparison  of  a  fowl 
protected  by  inoculation  with  the  microbe  of  fowl  cholera,  with  a  culture 
fluid  in  which  the  growth  of  a  particular  organism  has  exhausted  the  pabu- 
lum necessary  for  the  development  of  additional  organisms  of  the  same  kind, 
does  not  seem  to  me  to  be  a  just  one,  as  in  the  latter  case  we  have  a  limited 
supply  of  nutriment,  while  in  the  former  we  have  new  supplies  constantly 
provided  of  the  material — food — from  which  the  whole  body,  including  the 
hypothetical  substance  essential  to  the  development  of  the  disease  germ,  was 
built  up  prior  to  the  attack.  Besides  this  we  have  a  constant  provision  for 
the  elimination  of  effete  and  useless  products. 

"  This  hypothesis,  then,  requires  the  formation  in  the  human  body,  and 
the  retention  up  to  a  certain  time,  of  a  variety  of  materials  which,  so  far  as 
we  can  see,  serve  no  purpose  except  to  nourish  the  germs  of  various  specific 
diseases,  and  which,  having  served  this  purpose,  are  not  again  formed  in  the 
same  system,  subjected  to  similar  external  conditions,  and  supplied  with  the 
same  kind  of  nutriment." 

It  is  unnecessary  to  discuss  this  hypothesis  any  further,  inasmuch 
as  it  is  no  longer  sustained  by  Pasteur  or  his  pupils,  and  is  evidently 
untenable. 

The  Retention  Theory,  proposed  by  Chauveau  (1880),  is  subject  to 
similar  objections.  According  to  this  view,  certain  products  formed 
during  the  development  of  a  pathogenic  microorganism  in  the  body 
of  a  susceptible  animal  accumulate  during  the  attack  and  are  subse- 
quently retained,  and,  being  prejudicial  to  the  growth  of  the  particu- 
lar microorganism  which  produced  them,  a  second  infection  cannot 
occur.  Support  for  this  theory  has  been  found  by  its  advocates  in 
the  fact  that  various  processes  of  fermentation  are  arrested  after  a 
time  by  the  formation  of  substances  which  restrain  the  development 
<  >f  the  microorganisms  to  which  they  are  due.  But  in  the  case  of  a 
living  animal  the  conditions  are  very  different,  and  it  is  hard  to  con- 
ceive that  adventitious  products  of  this  kind  could  be  retained  for 
years,  when  in  the  normal  processes  of  nutrition  and  excretion  the 
tissues  and  fluids  of  the  body  are  constantly  undergoing  change. 
Certainly  the  substances  which  arrest  ordinary  processes  of  f ermen- 


SUSCEPTIBILITY   AND   IMMUNITY.  251 

tation  by  their  accumulation  in  the  fermenting  liquid,  such  as  alco- 
hol, lactic  acid,  phenol,  etc. ,  would  not  be  so  retained.  But  we  can- 
not speak  so  positively  with  reference  to  the  toxic  albuminous 
substances  which  recent  researches  have  demonstrated  to  be  present 
in  cultures  of  some  of  the  best  known  pathogenic  bacteria.  It  is 
difficult,  however,  to  believe  that  an  individual  who  has  passed 
through  attacks  of  half  a  dozen  different  infectious  diseases  carries 
about  with  him  a  store  of  as  many  different  chemical  substances  pro- 
duced during  these  attacks,  and  sufficient  in  quantity  to  prevent  the 
development  of  the  several  germs  of  these  diseases.  Nor  does  the 
experimental  evidence  relating  to  the  action  of  germicide  and  germ- 
restraining  agents  justify  the  view  that  a  substance  capable  of 
preventing  the  development  of  one  microorganism  should  be  with- 
out effect  upon  others  of  the  same  class ;  but  if  we  accept  the  re- 
tention hypothesis  we  must  admit  that  the  inhibiting  substance 
produced  by  each  particular  pathogenic  germ  is  effective  only  in 
restraining  the  development  of  the  microbe  which  produced  it  in  the 
first  instance. 

Pasteur  discusses  this  hypothesis  in  his  paper  from  which  we 
have  already  quoted,  as  follows  : 

4 '  We  may  admit  the  possibility  that  the  development  of  the  microbe,  in 
place  of  removing  or  destroying  certain  matters  in  the  bodies  of  the  fowls, 
adds,  on  the  contrary,  something  which  is  an  obstacle  to  the  future  develop- 
ment of  this  microbe.  The  history  of  the  life  of  inferior  beings  authorizes 
such  a  supposition.  The  excretions  resulting  from  vital  processes  may  arrest 
vital  processes  of  the  same  nature.  In  certain  fermentations  we  see  anti- 
septic products  make  their  appearance  during,  and  as  a  result  of,  the  fer- 
mentation, which  put  an  end  to  the  active  life  of  the  ferments  and  arrest 
the  fermentations  long  before  they  are  completed.  In  the  cultivation  of  our 
microbe,  products  may  have  been  formed  the  presence  of  which,  possibly, 
may  explain  the  protection  following  inoculation. 

"Our  artificial  cultures  permit  us  to  test  the  truth  of  this  hypothesis. 
Let  us  prepare  an  artificial  culture  of  the  microbe,  and  after  having  evapo- 
rated it,  in  vacuo,  without  heat,  let  us  bring  it  back  to  its  original  volume 
by  means  of  fresh  chicken  bouillon.  If  the  extract  contains  a  poison  for 
the  life  of  the  microbe,  and  if  this  is  the  cause  of  its  failure  to  multiply  in  the 
filtered  liquid,  the  new  liquid  should  remain  sterile.  Now,  this  is  not  the  case. 
We  cannot,  then,  believe  that  during  the  life  of  the  parasite  certain  substances 
are  produced  which  are  capable  of  arresting  its  ulterior  development." 

This  experiment  of  Pasteur  appears  to  be  conclusive  so  far  as  the 
particular  pathogenic  microorganism  referred  to  is  concerned ;  and 
we  may  say,  in  brief,  that  more  recent  investigations  do  not  sustain 
the  view  that  acquired  immunity  is  due  to  the  retention  of  products 
such  as  are  formed  by  pathogenic  bacteria  in  artificial  culture  media, 
and  which  act  by  destroying  these  bacteria  or  restraining  their  devel- 
opment when  they  are  introduced  into  the  bodies  of  immune  animals. 

Moreover,  if  we  suppose  that  the  toxic  substances  which  give 
pathogenic  power  to  a  particular  microorganism  are  retained  in  the 


252  SUSCEPTIBILITY   AND   IMMUNITY. 

body  of  an  immune  animal,  we  must  admit  that  the  animal  has  ac- 
quired a  tolerance  to  the  pathogenic  action  of  these  toxic  substances, 
for  their  presence  no  longer  gives  rise  to  any  morbid  phenomena. 
And  this  being  the  case,  we  are  not  restricted  to  the  explanation 
that  immunity  depends  upon  a  restraining  influence  exercised  upon 
the  microbe  when  subsequently  introduced. 

The  Vital  Resistance  Theory. — Another  explanation  offers  itself, 
viz.,  that  immunity  depends  upon  an  acquired  tolerance  to  the 
toxic  products  of  pathogenic  bacteria.  This  is  a  view  which  the 
writer  has  advocated  in  various  published  papers  since  1881.  In  a 
paper  contributed  to  the  American  Journal  of  the  Medical  Sci- 
ences in  April,  1881,  it  is  presented  in  the  following  language: 

"The  view  that  I  am  endeavoring-  to  elucidate  is  that,  during  a  non- 
fatal  attack  of  one  of  the  specific  diseases,  the  cellular  elements  implicated 
which  do  not  succumb  to  the  destructive  influence  of  the  poison  acquire  a 
tolerance  to  this  poison  which  is  transmissible  to  their  progeny,  and  which 
is  the  reason  of  the  exemption  which  the  individual  enjoys  from  future 
attacks  of  the  same  disease. " l 

In  my  chapter  on  "Bacteria  in  Infectious  Diseases,"  in  "Bac- 
teria," published  in  the  spring  of  1884,  but  placed  in  the  hands  of  the 
publishers  in  1883,  I  say: 

"  It  may  be  that  the  true  explanation  of  the  immunity  afforded  by  a  mild 
attack  of  an  infectious  germ  disease  is  to  be  found  in  an  acquired  tolerance  to 
the  action  of  a  chemical  poison  produced  by  the  microorganism,  and  conse- 
quent ability  to  bring  the  resources  of  nature  to  bear  to  restrict  invasion  by 
Uie  parasite." 

The  "resources  of  nature"  are  referred  to  in  the  same  chapter  as 
follows  : 

44  The  hypothesis  of  Pasteur  would  account  for  the  fact  that  one  individual 
suffers  a  severe  attack  and  another  a  mild  attack  of  an  infectious  disease, 
after  being  subjected  to  the  influence  of  the  poison  under  identical  circum- 
stances, by  the  supposition  that  the  pabulum  required  for  the  development 
of  this  particular  poison  is  more  abundant  in  the  body  of  one  individual 
than  in  the  other.  The  explanation  which  seems  to  us  more  satisfactory  is 
that  the  vital  resistance  offered  by  the  cellular  elements  in  the  bodies  of 
these  two  individuals  was  not  the  same  for  this  poison.  It  is  well  known 
that  in  conditions  of  lowered  vitality  resulting  from  starvation,  profuse 
discharges,  or  any  other  cause,  the  power  to  resist  disease  poisons  is  greatly 
diminished,  and,  consequently,  that  the  susceptibility  of  the  same  individual 
differs  at  different  times. 

44  From  our  point  of  view,  the  blood,  as  it  is  found  within  the  vessels  of  a 
living  animal,  is  not  simply  a  culture  fluid  maintained  at  a  fixed  tempera- 
ture, out  under  these  circumstances  is  a  tissue,  the  histological  elements  of 
which  present  a  certain  vital  resistance  to  pathogenic  organisms  which  may 
be  introduced  into  the  circulation. 

44  If  we  add  a  small  quantity  of  a  culture  fluid  containing  the  bacteria  of 
putrefaction  to  the  blood  of  an  animal,  withdrawn  from  the  circulation  into 
a  proper  receptacle  and  maintained  in  a  culture  oven  at  blood  heat,  we  will 
find  that  these  bacteria  multiply  abundantly,  and  evidence  of  putrefactive 

1  "What  is  the  Explanation  of  the  Protection  from  Subsequent  Attacks,  result- 
ing from  :m  Attack  of  Certain  Diseases,  etc  ?"  American  Journal  of  the  Medical 
Sciences,  April,  1881,  p.  370. 


SUSCEPTIBILITY   AND   IMMUNITY.  #53 

decomposition  will  soon  be  perceived.  But  if  we  inject  a  like  quantity  of 
the  culture  fluid  with  its  contained  bacteria  into  the  circulation  of  a  living- 
animal,  not  only  does  no  increase  and  no  putrefactive  change  occur,  but  the 
bacteria  introduced  quickly  disappear,  and  at  the  end  of  an  hour  or  two  the 
most  careful  microscopical  examination  will  not  reveal  the  presence  of  a 
single  bacterium.  This  difference  we  ascribe  to  the  vital  properties  of  the 
fluid  as  contained  in  the  vessels  of  a  living  animal;  and  it  seems  probable 
that  the  little  masses  of  protoplasm  known  as  white  blood  corpuscles  are  the 
essential  histological  elements  of  the  blood,  so  far  as  any  manifestation  of 
vitality  is  concerned.  The  ivriter  has  elsewhere  (1881)  suggested  that  the 
disappearance  of  the  bacteria  from  the  circulation,  in  the  experiment 
referred  to,  may  be  effected  by  the  white  corpuscles,  which,  it  is  well  known, 
pick  up,  after  the  manner  of  amosbae,  any  particles,  organic  or  inorganic, 
which  come  in  their  way.  And  it  requires  no  great  stretch  of  credulity  to 
believe  that  they  may,  like  an  amoeba,  digest  and  assimilate  the  protoplasm 
of  the  captured  bacterium,  thus  putting  an  end  to  the  possibility  of  its  do- 
ing any  harm. 

"  In  the  case  of  a  pathogenic  organism  we  may  imagine  that,  when  cap- 
tured in  this  way,  it  may  share  a  like  fate  if  the  captor  is  not  paralyzed  by 
some  potent  poison  evolved  by  it,  or  overwhelmed  by  its  superior  vigor  and 
rapid  multiplication.  In  the  latter  event  the  active  career  of  our  conserva- 
tive white  corpuscle  would  be  quickly  terminated  and  its  protoplasm  would 
serve  as  food  for  the  enemy.  It  is  evident  that  in  a  contest  of  this  kind  the 
balance  of  power  would  depend  upon  circumstances  relating  to  the  inherited 
vital  characteristics  of  the  invading  parasite  and  of  the  invaded  leucocyte." 

In  the  same  chapter  the  writer  quotes  from  his  paper  on  acquired 
immunity,  published  in  1881,  as  follows  : 

"  The  difficulties  into  which  this  hypothesis  [the  exhaustion  theory  of  Pas- 
teur] leads  us  certainly  justify  us  in  looking  further  for  an  explanation  of  the 
phenomena  in  question.  This  explanation  is,  I  believe,  to  be  found  in  the 
peculiar  properties  of  the  protoplasm,  which  is  the  essential  framework  of 
every  living  organism.  The  properties  referred  to  are  the  tolerance  which 
living  protoplasm  may  acquire  to  certain  agents  which,  in  the  first  instance, 
have  an  injurious  or  even  fatal  influence  upon  its  vital  activity  ;  and  the 
property  which  it  possesses  of  transmitting  its  peculiar  qualities,  inherent  or 
acquired,  through  numerous  generations,  to  its  offshoots  or  progeny. 

"Protoplasm  is  the  essential  living  portion  of  the  cellular  elements  of  ani- 
mal and  vegetable  tissues ;  but  as  our  microscopical  analysis  of  the  tissues  has 
not  gone  beyond  the  cells  of  which  they  are  composed,  and  is  not  likely  to 
reveal  to  us  the  complicated  molecular  structure  of  the  protoplasm,  upon 
which,  possibly,  the  properties  under  consideration  depend,  it  will  be  best, 
for  the  present,  to  limit  ourselves  to  a  consideration  of  the  living  cells  of  the 
body.  These  cells  are  the  direct  descendants  of  the  pre-existent  cells,  and 
may  all  be  traced  back  to  the  sperm  cell  and  the  germ  cell  of  the  parents. 
Now,  the  view  which  I  am  endeavoring  to  elucidate  is  that,  during  a  non- 
fatal  attack  of  one  of  the  specific  diseases,  the  cellular  elements  implicated, 
which  do  not  succumb  to  the  destructive  influence  of  the  poison,  acquire  a 
tolerance  to  this  poison  which  is  transmissible  to  their  progeny,  and  which 
is  the  reason  of  the  exemption  which  the  individual  enjoys  from  future 
attacks  of  the  same  disease. 

"  The  known  facts  in  regard  to  the  hereditary  transmission  by  cells  of  ac- 
quired properties  make  it  easy  to  believe  in  the  transmission  of  such  a 
tolerance  as  we  imagine  to  be  acquired  during  the  attack ;  and  if  it  is  shown 
by  analogy  that  there  is  nothing  improbable  in  the  hypothesis  that  such  a 
tolerance  is  acquired,  we  shall  have  a  rational  explanation,  not  of  heredity 
and  of  the  mysterious  properties  of  protoplasm,  but  of  the  particular  result 
under  consideration.  The  transmission  of  acquired  properties  is  shown  in 
the  budding  and  grafting  of  choice  fruits  and  flowers,  produced  by  cultiva- 


254  SUSCEPTIBILITY   AND   IMMUNITY, 

tion,  upon  the  wild  stock  from  which  they  originated.  The  acquired  proper- 
ties are  transmitted  indefinitely;  and  the  same  sap  which  on  one  twig  nour- 
ishes a  sour  crab  apple,  on  another  one  of  the  same  branch  is  elaborated  into 
a  delicious  pippin. 

"  The  tolerance  to  narcotics — opium  and  tobacco — and  to  corrosive  poisons 
— arsenic — which  results  from  a  gradual  increase  of  dose,  may  be  cited  as  an 
example  of  acquired  tolerance  by  living  protoplasm  to  poisons  which  at  the 
outset  would  have  been  fatal  in  much  smaller  doses. 

"The  immunity  which  an  individual  enjoys  from  any  particular  disease 
must  be  looked  upon  as  a  power  of  resistance  possessed  by  the  cellular  ele- 
ments of  those  tissues  of  his  body  which  would  yield  to  the  poison  in  the 
case  of  an  unprotected  person." 

This  theory  of  immunity,  advanced  by  the  author  in  1881,  has 
received  considerable  support  from  investigations  made  since  that 
date,  and  especially  from  the  experimental  demonstration  by  Sal- 
mon, Roux,  and  others  that,  as  suggested  in  the  paper  from  which  I 
have  quoted,  immunity  may  result  from  the  introduction  into  the 
body  of  a  susceptible  animal  of  the  soluble  products  of  bacterial 
growth — filtered  cultures. 

The  theory  of  vital  resistance  to  the  toxic  products  evolved  by 
pathogenic  bacteria  is  also  supported  by  numerous  experiments 
which  show  that  natural  or  acquired  immunity  may  be  overcome 
when  these  toxic  products  are  introduced  in  excess,  or  when  the  vital 
resisting  power -of  the  animal  has  been  reduced  by  various  agencies. 

More  direct  experimental  evidence  in  favor  of  the  view  under  con- 
sideration is  that  obtained  by  Beumer  in  his  experiments  with  steril- 
ized cultures  of  the  typhoid  bacillus.  He  found  that  after  the  re- 
peated injection  of  non-lethal  doses  mice  were  able  to  resist  an 
amount  of  this  toxine  which  was  fatal  to  animals  of  the  same  spe- 
cies not  so  treated.  But,  on  the  other  hand,  Gamaleia  found,  in  his 
experiments  upon  guinea-pigs  which  had  been  made  immune  against 
the  pathogenic  action  of  a  spirillum,  called  by  him  Vibrio  Metschni- 
kovi,  that  these  animals  have  no  increased  tolerance  for  the  toxic 
products  of  this  microorganism.  Although  immune  against  infec- 
tion by  the  living  microbe,  they  were  killed  by  the  same  quantity  of 
a  sterilized  culture  as  was  fatal  to  guinea-pigs  which  had  not  been 
rendered  immune. 

Charrin  has  obtained  similar  results  in  experiments  with  filtered 
cultures  of  Bacillus  pyocyaneus.  Rabbits  which  had  an  artificial  im- 
munity against  the  pathogenic  action  of  the  bacillus  were  killed  by 
doses  of  a  sterilized  culture  such  as  were  fatal  to  other  rabbits  of  the 
same  size  not  immune.  In  subsequent  experiments  by  Charrin  and 
Gameleia  "vaccinated"  rabbits  were  found  to  be  even  more  suscepti- 
ble to  the  toxic  action  of  filtered  cultures  than  were  those  not  vacci- 
nated. Metschnikoff  (1891)  has  followed  up  this  line  of  experiment, 
and  has  shown  that  when  considerable  amounts  of  filtered  cultures 
of  Bacillus  pyocyaneus  are  injected  subcutaneously  in  rabbits  a  cer- 


SUSCEPTIBILITY   AND   IMMUNITY.  255 

tain  tolerance  to  the  toxic  action  of  the  same  cultures  is  established 
in  some  instances.  But  his  results  do  not  give  any  substantial  sup- 
port to  the  view  that  immunity  depends  upon  an  acquired  tolerance 
to  the  toxic  action  of  the  chemical  products  contained  in  cultures  of 
the  pathogenic  bacteria  with  which  he  experimented — Bacillus  pyo- 
cyaneus  and  Vibrio  Metschnikovi. 

In  view  of  the  results  of  experimental  researches  above  recorded, 
and  of  other  recent  experiments  which  show  that,  in  certain  cases  at 
least,  acquired  immunity  depends  upon  the  formation  of  an  anti- 
fcoxine  in  the  body  of  the  immune  animal,  we  are  convinced  that  the 
theory  of  immunity  under  discussion,  first  proposed  by  the  writer  in 
1881,  cannot  be  accepted  as  a  sufficient  explanation  of  the  facts  in 
general.  At  the  same  time  we  are  inclined  to  attribute  considerable 
importance  to  acquired  tolerance  to  the  toxic  products  of  pathogenic 
bacteria  as  one  of  the  factors  by  which  recovery  from  an  infectious 
disease  is  made  possible  and  subsequent  immunity  established. 

The  "  vital-resistance  theory"  of  the  present  writer,  as  set  forth 
in  the  above-quoted  extracts  from  his  published  papers,  is  essentially 
the  same  as  that  advocated  by  Buchner  at  a  later  date  (1883).  Buch- 
ner  supposes  that  during  the  primary  infection,  when  an  animal  re- 
covers, a  "  reactive  change  "  has  been  produced  in  the  cells  of  the 
body  which  enables  it  to  protect  itself  from  the  pathogenic  action 
of  the  same  microorganism  when  subsequently  introduced. 

Of  course  when  we  ascribe  immunity  to  the  "  vital  resistance"  of 
the  cellular  elements  of  the  body,  we  have  not  explained  the 
modus  operandi  of  this  vital  resistance  or  "  reactive  change,"  but 
have  simply  affirmed  that  the  phenomenon  in  question  depends  upon 
some  acquired  property  residing  in  the  living  cellular  elements  of 
the  body.  We  have  suggested  that  that  which  has  been  acquired 
is  a  tolerance  to  the  action  of  the  toxic  products  produced  by  patho- 
genic bacteria.  But,  as  already  stated,  in  the  light  of  recent  experi- 
ments this  theory  now  appears  to  us  to  be  untenable  as  a  general 
explanation  of  acquired  immunity. 

The  Theory  of  Phagocytosis. — The  fact  that  in  certain  infectious 
diseases  due  to  bacteria  the  parasitic  invaders,  at  the  point  of  inocu- 
lation or  in  the  general  blood  current,  are  picked  up  by  the  leuco- 
cytes and  in  properly  stained  preparations  may  be  seen  in  their  in- 
terior, has  been  known  for  some  years.  In  mouse  septicaemia — an 
infectious  disease  described  by  Koch  in  his  work  on  "Traumatic 
Infectious  Diseases, "  published  in  1878 — the  slender  bacilli  which  are 
the  cause  of  the  disease  are  found  in  large  numbers  in  the  interior  of 
the  leucocytes.  Koch  says,  in  the  work  referred  to  :  "  Their  rela- 
tion to  the  white  blood  corpuscles  is  peculiar  ;  they  penetrate  these 
and  multiply  in  their  interior.  One  often  finds  that  there  is 


256  SUSCEPTIBILITY   AND    IMMUNITY. 

hardly  a  single  white  corpuscle  in  the  interior  of  which  bacilli  can- 
not be  seen.  Many  corpuscles  contain  isolated  bacilli  only ;  others 
have  thick  masses  in  their  interior,  the  nucleus  being  still  recog- 
nizable ;  while  in  others  the  nucleus  can  be  no  longer  distinguished  ; 
and,  finally,  the  corpuscle  may  become  a  cluster  of  bacilli,  breaking 
up  at  the  margin — the  origin  of  which  one  could  not  have  explained 
had  there  been  no  opportunity  of  seeing  all  the  intermediate  steps 
between  the  intact  white  corpuscle  and  these  masses  "  (Fig.  78).  It 
will  be  noted  that  in  the  above,  quotation  Koch  affirms  that  the 
bacilli  penetrate  the  leucocytes  and  multiply  in  their  interior.  Now, 
the  theory  of  phagocytosis  assumes  that  the  bacilli  are  picked  up  by 
the  leucocytes  and  destroyed  in  their  interior,  and  that  immunity  de- 
pends largely  upon  the  power  of  these  "  phagocytes"  to  capture  and 
destroy  living  pathogenic  bacilli. 

The  writer  suggested  this  as  an  hypothesis  as  long  ago  as  1881, 
in  a  paper  read  before  the  American  Association  for  the  Advance- 
ment of  Science,  in  the  following  language: 

"It  has  occurred  to  me  that  possibly  the  white  corpuscles  may 
have  the  office  of  picking  up  and  digesting  bacterial  organisms  which 


FIG.  78.— Bacillus  of  mouse  septicaemia  in  leucocytes  from  blood  of  mouse  (Koch). 

by  any  n^eans  find  their  way  into  the  blood.  The  propensity  exhib- 
ited by  the  leucocytes  for  picking  up  inorganic  granules  is  well 
known,  and  that  they  may  be  able  not  only  to  pick  up  but  to  assimi- 
late, and  so  dispose  of,  the  bacteria  which  come  in  their  way,  does 
not  seem  to  me  very  improbable,  in  view  of  the  fact  that  amoebae, 
which  resemble  them  so  closely,  feed  upon  bacteria  and  similar  or- 
ganisms." ' 

At  a  later  date  (1884)  Metschnikoff  offered  experimental  evi- 
dence in  favor  of  this  view,  and  the  explanation  suggested  in  the 
above  quotation  is  commonly  spoken  of  as  the  Metschnikoff  theory. 

1  "  A  Contribution  to  the  Study  of  Bacterial  Organisms  commonly  found  upon 
Exposed  Mucous  Surfaces  and  in  the  Alimentary  Canal  of  Healthy  Individuals."  Il- 
lustrated by  photomicrographs.  Proceedings  of  the  American  Association  for  Ad- 
vancement  of  Science,  1881,  Salem,  1882,  xxx.,  83-94.  Also  in  Studies  from  the 
Biological  Laboratory,  Johns  Hopkins  University,  Baltimore,  vol.  ii.,  No.  2,  1882. 


SUSCEPTIBILITY    AND    IMMUNITY.  257 

The  observations  which  first  led  Metschnikoff  to  adopt  this  view 
were  made  upon  a  species  of  daphnia  which  is  subject  to  fatal  infec- 
tion by  a  torula  resembling  the  yeast  fungus.  Entering  with  the 
food,  this  fungus  penetrates  the  walls  of  the  intestine  and  invades  the 
tissues.  In  certain  cases  the  infection  does  not  prove  fatal,  owing,  as 
Metschnikoff  asserts,  to  the  fact  that  the  fungus  cells  are  seized  upon 
by  the  leucocytes,  which  appear  to  accumulate  around  the  invading 
parasite  (chemiotaxis)  for  this  special  purpose.  If  they  are  success- 
ful in  overpowering  and  destroying  the  parasite  the  animal  recovers ; 
if  not,  it  succumbs  to  the  general  infection  which  results.  In  a  simi- 
lar manner,  Metschnikoff  supposes,  pathogenic  bacteria  are  destroyed 
when  introduced  into  the  body  of  an  immune  animal.  The  colorless 
blood  corpuscles,  which  he  designates  phagocytes,  accumulate  at  the 
point  of  invasion  and  pick  up  the  living  bacteria,  as  they  are  known 
to  pick  up  inorganic  particles  injected  into  the  circulation.  So  far 
there  can  be  no  doubt  that  Metschnikoff  is  right.  The  presence  of 
bacteria  in  the  leucocytes  in  considerable  numbers,  both  at  the  point 
of  inoculation  and  in  the  general  circulation,  has  been  repeatedly 
demonstrated  in  animals  inoculated  with  various  pathogenic  bacteria. 
The  writer  observed  this  in  his  experiments,  made  in  1881,  in  which 
rabbits  were  inoculated  with  cultures  of  his  Micrococcus  Pasteuri ; 
and  it  was  this  observation  which  led  him  to  suggest  the  theory 
which  has  since  been  so  vigorously  supported  by  Metschnikoff.  But 
the  presence  of  a  certain  number  of  bacteria  within  the  leucocytes 
does  not  prove  the  destructive  power  of  these  cells  for  living  patho- 
genic organisms.  As  urged  by  Weigert,  Baumgarten,  and  others, 
it  may  be  that  the  bacteria  were  already  dead  when  they  were  picked 
up,  having  been  destroyed  by  some  agency  outside  of  the  blood  cells. 
As  heretofore  stated,  we  have  now  experimental  evidence  that  blood 
serum,  quite  independently  of  the  cellular  elements  contained  in  it 
in  the  circulation,  has  decided  germicidal  power  for  certain  patho- 
genic bacteria,  and  that  the  blood  serum  of  the  rat  and  other  animals 
which  have  a  natural  immunity  against  anthrax  is  especially  fatal 
to  the  anthrax  bacillus. 

Numerous  experiments  have  been  made  during  the  past  two  or 
three  years  with  a  view  to  determining  whether  pathogenic  bacteria 
are,  in  fact,  destroyed  within  the  leucocytes  after  being  picked  up, 
and  different  experimenters  have  arrived  at  different  conclusions. 
In  the  case  of  mouse  septicaemia,  already  alluded  to,  and  in  gonor- 
rhoea, one  would  be  disposed  to  decide,  from  the  appearance  and  ar- 
rangement of  the  pathogenic  bacteria  in  the  leucocytes,  that  they  are 
not  destroyed,  but  that,  on  the  other  hand,  they  multiply  in  the  in- 
terior of  these  cells,  which  in  the  end  succumb  to  this  parasitic  in- 
vasion. In  both  of  the  diseases  mentioned  we  find  leucocytes  so 
17 


258  SUSCEPTIBILITY   AND   IMMUNITY. 

completely  filled  with  the  pathogenic  microorganisms  that  it  is  diffi- 
cult to  believe  that  they  have  all  been  picked  up  by  a  voracious  pha- 
gocyte, which  has  stuffed  itself  to  repletion,  while  numerous  other 
leucocytes  from  the  same  source  and  in  the  same  microscopic  field  of 
view  have  failed  to  capture  a  single  bacillus  or  micrococcus.  More- 
over, the  staining  of  the  parasitic  invaders,  and  the  characteristic  ar- 
rangement of  the  ' '  gonococcus  "  in  stained  preparations  of  gonorrhoeal 
pus,  indicate  that  their  vitality  has  not  been  destroyed  in  the  interior 
of  the  leucocytes  or  pus  cells,  and  we  can  scarcely  doubt  that  the 
large  number  found  in  certain  cells  is  due  to  multiplication  in  situ 
rather  than  to  an  unusual  activity  of  these  particular  cells.  But  in 
certain  infectious  diseases,  and  especially  in  anthrax,  the  bacilli  in- 
cluded within  the  leucocytes  often  give  evidence  of  degenerative 
changes,  which  would  support  the  view  that  they  are  destroyed  by 
the  leucocytes,  unless  these  changes  occurred  before  they  were  picked 
up,  as  is  maintained  by  Nuttall  and  others.  We  cannot  consider 
this  question  as  definitely  settled,  but,  in  view  of  the  importance 
attached  to  the  theory  of  phagocytosis  by  many  pathologists  and  bac- 
teriologists, we  reproduce  here  a  paper  by  Metschnikoff  in  which  his 
views  are  fully  set  forth  : 

LECTURE  ON  PHAGOCYTOSIS  AND   IMMUNITY.1 

It  is  not  possible  to  study  the  bacteriology  of  disease  without  noticing 
that,  while  ^n  many  cases  the  invading  microorganisms  are  to  be  found 
solely  in  the  fluids  of  the  body,  in  not  a  few  affections  they  present  them- 
selves in  the  interior  of  certain  cells,  and  this  either  partially — some  being 
within  the  cells,  others  free  in  the  blood  plasma  and  the  lymph  that  bathes 
the  various  tissues — or  exclusively,  all  the  bacteria  that  are  visible  being 
intracellular.  Many  of  the  facts  bearing  upon  the  terms  of  this  relationship 
between  tissue  cell  and  microorganism  are  now  well  known,  yet  it  is  worth 
while  to  recapitulate  the  more  important,  in  order  to  show  that  from  them  it 
is  possible  to  gain  a  general  law  ;  and  what  is  more,  that  from  a  study  of 
such  facts  some  insight  may  be  gained  into  the  phenomena  of  immunity. 

It  may,  in  the  first  place,  be  postulated  that  whenever  a  microorganism 
is  discoverable  within  a  cell  its  passage  thither  has  been  by  means  of  proto- 
plasmic or  amoeboid  movements,  either  on  the  part  of  the  microbe  or  of  the 
cell  itself.  The  first  alternative  is  the  rarer,  although  it  certainly  exists,  and 
of  this  the  malarial  parasite  affords  an  excellent  example ;  for  here  in  the 
amoeboid  stage  of  its  existence  the  hsematozoon  makes  its  way  into  the  in- 
trrior  of  a  cell  that  possesses  no  active  movements  of  its  own,  namely,  the 
red  blood  corpuscle,  and  from  the  substance  of  this  corpuscle  the  parasite 
gains  its  nourishment.  Other  sporozoa  furnish  instances  almost  equally 
good.  More  commonly,  however,  as  in  the  case  of  all  bacteria,  where  we 
have  to  deal  wit  h  microorganism*  which,  even  when  mobile,  are  destitute  of 
protoplasmic  appendages,  it  is  the  cells  which  play  the  active  part ;  certain 
cells  include  the  parasites.  Of  such  the  amoebifonn  leucocyte  of  the  blood 
and  lymph  is  the  most  typical  example,  capable,  as  it  is,  of  sending  out 
pseudopodia  in  all  directions,  while  a  closely  allied  form  is  the  cell  of  the 

1  Delivered  at  the  Institut  Pasteur,  December  29th,  1890,  by  Dr.  Elias  Metschni- 
koff, Chef  de  Service  do  1' Institut  Pasteur,  Paris  ;  late  Professor  of  Zoology  in  the 
University  of  Odessa. 


SUSCEPTIBILITY   AND    IMMUNITY.  259 

splenic  pulp.  But  there  are  also  cells — as,  for  instance,  those  forming  the 
endothelial  lining-  of  the  vessels — which  are  very  definitely  fixed,  which 
nevertheless  can  give  off  protoplasmic  processes  from  their  free  surface  and 
so  capture  and  include  bacteria. 

All  these  may  be  spoken  of  as  phagocytes,  and  may  be  divided  into  the 
two  broad  groups  affixed  phagocytes  (endothelial  cells,  etc.)  and  free  (leu- 
cocytes). Not  that  the  terms  "phagocyte"  and  "leucocyte"  are  synonymous, 
for  of  the  latter  three  main  forms  may  be  distinguished,  of  which  one  is 
practically  immobile  and  never  takes  up  bacteria.  This  is  the  lymphocyte, 
characterized  by  its  relatively  small  size,  its  large  single  nucleus,  and  the 
small  amount  of  surrounding  protoplasm.  The  two  remaining  (phagocytic) 
forms  are,  first,  the  large  uninuclear  leucocyte,  whose  prominent  nucleus  is 
at  times  lobed  or  reniform,  which  stains  well  with  aniline  dyes  and  possesses 
much  protoplasm  and  active  amoeboid  movements — the  macrophage — and, 
second,  the  microphage,  a  small  form,  also  staining  well,  but  either  multi- 
nuclear  or  with  one  nucleus  in  the  process  of  breaking  up.  If  now  we  com- 
pare the  endothelial  cells  with  these,  it  is  evident  that  their  properties  con- 
nect them  closely  with  the  macrophage  ;  and,  in  fact,  there  is  now  little  or 
no  doubt  that  a  very  large  proportion  of  the  macrophages  are  of  endothelial 
origin. 

Leaving  aside  the  subject  of  amoeboid  microbes  and  their  life  within  ani- 
mal cells,  it  is  to  the  phagocytes  and  their  relation  to  the  bacteria  that  I  wish 
specially  to  draw  your  attention. 

Taking  as  wide  a  view  as  possible  of  this  relationship,  we  can  first  deter- 
mine that  the  more  malignant  the  microorganism  the  rarer  is  its  presence 
within  the  phagocyte.  Thus  in  those  which  of  all  diseases  are  the  most 
rapidly  fatal — in  chicken  cholera  affecting  birds  and  rabbits,  in  hog  cholera 
("cholera  des  pores")  given  to  pigeons  and  rabbits,  in  the  anthrax  of  mice 
and  other  specially  sensitive  animals,  in  the  "septicemie  vibrionienne"  of 
guinea-pigs  and  birds,  and  in  yet  other  diseases  of  peculiarly  swift  course — 
the  corresponding  bacteria  are  only  very  exceptionally  to  be  found  within 
the  cells,  but  remain  free  in  the  neighborhood  of  their  introduction  and 
thence  invade  the  blood.  For  all  the  above-mentioned  diseases  are  not 
localized,  but,  on  the  contrary,  present  the  characters  of  general  acute  sep- 
ticaemia, causing  death  within  twenty  to  thirty-six  hours,  or,  in  certain 
cases,  even  within  six  hours. 

And  when  we  pass  to  those  diseases  in  which  the  bacteria  are  to  be  found 
either  in  part  or  almost  wholly  within  the  phagocytes,  the  same  law  still 
applies  ;  for  in  such  cases  the  disease  has  lost  its  suddenness,  tending  to 
have  a  slower  course,  or,  indeed,  to  be  of  a  chronic  nature.  Even  in  those 
affections  in  which  an  acute  course  is  accompanied  by  considerable  phago- 
cytosis, the  fatal  termination  is  far  from  occurring  at  the  same  early  period 
as  in  the  diseases  recorded  above.  Thus  mouse  septicaemia,  characterized  as 
it  is  by  frequent  intracellular  bacteria,  has  a  duration  in  the  mouse  two  and 
a  half  times  as  long  as  that  of  anthrax  in  the  same  animal.  But  in  general  a 
well-marked  phagocytosis  is  associated  with  diseases  presenting  an  essen- 
tially chronic  development ;  it  is  in  affections  such  as  tuberculosis,  leprosy, 
rhinoscleroma,  glanders,  that  the  specific  bacteria  are  most  readily  taken  up 
by  the  phagocytes ;  it  is  here  that,  at  the  seat  of  the  disease,  we  meet  with  in- 
numerable macrophages — epithelioid  cells  in  which  lie  the  individual  micro- 
organisms. 

Further,  if  we  consider  the  phenomena  associated  with  the  resolution  of 
an  infectious  disease,  this  inverse  relationship  between  the  malignancy  of  the 
malady  and  the  occurrence  of  phagocytosis  is,  if  possible,  yet  more  clearly 
demonstrated.  Notice,  for  instance,  what  obtains  during  the  progress  of  re- 
lapsing fever,  a  malady  still  fairly  common  in  Russia  and  other  Sclavonic 
countries,  and  one  which,  while  presenting  many  difficulties  to  the  bacteri- 
ologist, in  that  the  specific  spirochaete  has  so  far  resisted  cultivation,  and  in 
that  it  cannot  be  communicated  to  the  ordinary  animals  of  the  laboratory,  is 
nevertheless  in  many  respects  not  ill -adapted  for  our  present  purpose.  Here, 


260  SUSCEPTIBILITY   AND  IMMUNITY. 

during  the  sudden  access  of  the  fever,  the  spirilla  are  present  in  the  blood  in, 
enormous  numbers ;  they  all  are  free  in  the  plasma,  and  not  a  single  intra- 
cellular  spirillum  is  to  be  met  with.  During  the  apyretic  stage  (and  in  the 
monkey  this  is,  at  the  same  time,  the  stage  of  resolution)  not  a  single  free  spiril- 
lum is  discoverable  in  the  blood,  while  the  phagocytes  of  the  spleen  contain 
the  microbes.  The  like  phenomena  repeat  themselves  in  all  those  cases  where 
it  is  possible  to  follow  the  fate  of  the  microorganisms  of  acute  disease  during 
the  stage  of  recovery.  Thus  rats  and  pigeons  very  frequently  survive  an 
attack  of  anthrax,  and,  where  this  occurs,  the  bacteria,  which  ,at  the  com- 
mencement of  the  disease  were  for  the  most  part  free,  now,  during  resolution, 
are  for  the  most  part  included  within  leucocytes  and  splenic  phagocytes. 

Nor  is  this  all.  Analogous  phenomena  as  a  rule  attend  immunity,  which 
most  often  is  but  recovery  in  operation  from  the  very  onset  of  a  disease 
The  more  closely  one  studies  this  condition  of  immunity  the  more  is  one  led 
to  the  conviction  that  immunity  and  recovery  are  very  intimately  con- 
nected ;  that  one  can  pass  by  slight  gradations  from  the  resolution  of  disease 
to  the  production  of  immunity.  So  it  is  that,  in  inoculating  refractory  ani- 
mals with  the  microbe  to  whose  action  they  have  been  rendered  immune,  it 
is  found  that  the  parasite  begins  to  develop,  but  that  from  the  onset  a  reac- 
tion on  the  part  of  the  organism  shows  itself,  accompanied  by  a  considerable 
emigration  of  leucocytes,  which  soon  include  the  bacteria  in  great  numbers. 
This  relationship  of  phagocytosis  to  acquired  immunity  is  in  the  highest 
degree  instructive.  Where  a  given  species  of  animal  is  specially  sensitive 
to  the  onslaught  of  one  or  other  microorganism,  there,  during  the  course  of 
the  disease,  the  phagocvtes  are  inoperative,  including  none,  or  almost  none, 
of  the  bacteria.  On  the  other  hand,  when  by  previous  vaccination  these 
animals  have  been  rendered  refractory,  their  phagocytes  have  acquired  the 
property  of  including  the  same  bacteria.  As  an  example  of  this  I  may  cite 
the  action  of  the  bacillus  of  anthrax  and  of  the  Vibrio  Metschnikovi.  In 
ordinarv  rabbits  the  development  of  anthrax  is  only  followed  by  a  very 
feeble  phagocytosis,  while  in  vaccinated  rabbits  this  phagocytosis  is  very  ex- 
tensive. Corresponding  but  yet  more  strongly  marked  differences  are  to  be 
made  out  between  the  unvaccinated  guinea-pig — an  animal  most  readily 
affected  by  the  vibrionic  septicaemia — and  the  guinea-pig  vaccinated  against 
the  same;  after  inoculation  with  the  Vibrio  Metschnikovi  none  of  the  vibrios 
are  to  be  found  in  the  cells  of  the  former;  in  the  latter  the  phagocytes  are 
simply  replete  with  the  microbes. 

The  facts  enumerated  thus  far  would  seem  to  prove  that  there  exists  a 
certain  antagonism  between  ihe  microbes  and  the  phagocytes,  and  this  view 
is  confirmed  by  the  fact  that  in  general  the  microbes  find  the  interior  of  the 
phagocytes  an  unfavorable  medium  for  their  development  and  continued 
existence.  Very  often  it  is  possible  to  determine  absolutely  that  the  parasites 
are  killed  within  the  phagocytes;  after  inoculating  refractory  animals  with 
bacteria,  an  afflux  of  white  corpuscles  toward  the  region  of  inoculation  fol- 
lowed by  the  inclusion  of  the  bacteria  and  by  their  death,  is  seen  to  occur. 
'•-;  >'•'-"•>  can  be  well  followed  where  the  anthrax  bacilli  are  taken  into 
te  phagocytes  of  animals  that  are,  or  have  been  rendered,  immune  They 
occur  also  with  a  long  series  of  other  microorganisms  studied  in  thisconnec- 
tion,  and,  among  others,  m  the  case  of  the  tubercle  bacillus  invading  animals 
that  are  more  or  less  resistant.  The  giant  cells  of  tuberculosis  are,  in  fact 
huge  muUinuclear  phagocytes,  and  here  the  intracellular  destruction  of  the 
the  more  clearly  demonstrable,  inasmuch  as  the  microorganisms 
«>x  In  bit  such  very  evident  signs  of  degeneration;  the  bacilli  swell  their  en- 
veloping membrane  becomes  much  thickened  and  highly  refractive  and  in 
imio  the .content*  lose  their  power  of  fixing  the  stain  in*  material  so  tint 
eventually  nothing  is  left  Vut  slightly  yellowish  form's  rec^Whi  pro- 
!""•!""'- •""!  I"-"""',  tlm  Pillared  burilli;  ami  ihrsr  shadowy  bodies  unite 

rerbetg^^ 


SUSCEPTIBILITY   AND   IMMUNITY.  261 

tures  or  in  caseating  masses — these  changes  may  well  be  regarded  as  due  to 
a  specific  action  upon  the  part  of  the  giant  cells. 

The  broad  fact  that  the  invasion  of  the  organism  by  microbes  most  often 
induces,  on  the  one  hand,  an  inflammatory  reaction  with  its  associated  emi- 
gration of  leucocytes,  and  that,  on  the  other  hand,  the  phagocytes  are 
capable  of  including  and  destroying  the  invaders,  leads  us  to  admit  that  the 
afflux  of  phagocytes  to  the  invaded  region,  and  their  bactericidal  properties, 
are  mechanisms  which  serve  to  ward  off  bacterial  attack  and  to  maintain  the 
integrity  of  the  organism.  Where  the  phagocytes  do  not,  either  immediately 
or  eventually,  intervene,  but  leave  the  field  free  to  the  microbes,  these  last 
multiply  without  hindrance  and  succeed  in  killing  the  animal  within,  it  may 
be,  an  excessively  short  period.  Thus  the  microorganism  of  hog  cholera, 
which  is  left  quite  untouched,  kills  the  pigeon  in  the  course  of  a  few  hours 
— often  within  five  hours  after  inoculation ;  chicken  cholera  kills  not  only 
pigeons  but  also  rabbits  in  an  equally  short  period.  In  other  diseases  in 
which  the  phagocytes  appear  upon  the  scene  in  relatively  large  numbers, 
and  even  include  the  microorganisms,  the  latter  gain  the  day  whenever  and 
wherever  the  phagocytes  are  incapable  of  destroying  them  or  of  preventing 
their  growth. 

This  manifest  bactericidal  action  is  to  be  compared  with  the  phenomena 
of  intracellular  digestion  characteristic  of  amoeboid  cells  in  general,  and  of 
leucocytes  and  other  microbic  phagocytes  in  particular.  These  cells  have 
the  power  of  digesting  with  ease  red  corpuscles  and  other  organized  ele- 
ments, just  as  have  the  amoebae  proper  and  other  protozoa.  Among  these 
last  are  many  which  have  been  found  to  include  and  transform  bacteria  in 
exactly  the  same  way  as  do  the  phagocytes  of  the  higher  animals. 

Now,  in  determining  the  intervention  or  non-intervention  of  the  leuco- 
cytes in  this  war  between  the  organism  and  the  bacteria,  a  very  great  part 
is  played  by  the  sensitiveness  of  these  cells  to  external  influences,  and  es- 
pecially to  the  chemical  composition  of  their  environment.  The  leucocytes 
are  powerfully  attracted  by  many  microorganisms  and  the  resultants  of 
their  growth,  and  as  powerfully  repelled  by  others  and  their  resultants,  or, 
as  it  is  expressed,  they  have  a  positive  chemiotaxis  for  certain  microbes,  a 
negative  chemiotaxis  for  others.  The  existence  of  these  chemiotactic  pro- 
perties has  been  so  clearly  proved  of  late  by  the  researches  of  Leber,  Mas- 
sart  and  Bordet,  and  Gabritschevski  that  I  need  not  enter  into  a  fuller  ex- 
planation of  the  subject  here.  Where  negative  chemiotaxis  manifests  itself, 
there,  being  shunned  by  the  white  corpuscles,  the  parasites  freely  propagate 
themselves  and  induce  the  death  of  their  host.  Nevertheless  this  chemio- 
taxis is  not  immutable,  and  the  cells  can  become  accustomed  to  substances 
from  which  they  shrank  at  first — a  negative  may  thus  be  transformed  into  a 
positive  chemiotactic  state.  Such  obtains  in  acquired  immunity  ;  the  cells 
which  in  the  unvaccinated  animal  never  included  the  bacteria,  now  in  the 
vaccinated  take  them  up  readily.  .  .  . 

There  is  not  a  single  portion  of  the  theory  which  I  have  just  expounded 
but  has  encountered  a  lively  opposition.  Even  the  fundamental  fact  that 
the  phagocytes  are  capable  of  including  the  microbes  has  had  doubts  thrown 
upon  it ;  it  has  been  held  that  the  latter  insinuate  themselves  into  the  for- 
mer. Only  after  successive  series  of  observations  upon  the  phagocytes  and 
the  living  microbes  has  it  been  proved  that  assuredly  it  is  the  phagocytes 
which,  by  the  aid  of  their  pseudopodia,  themselves  include  the  microorgan- 
isms. The  observer  can  see  the  whole  process  in  the  case  of  immobile  ba- 
cilli— can  see  the  leucocyte  approach,  send  out  pseudopodia,  arid  gradually 
include  the  individual  bacillus.  Or,  conversely,  in  cases  of  negative  che- 
miotaxis, one  can,  in  blood  taken  from  the  monkey  during  the  access  of  re- 
lapsing fever,  observe  the  actively  moving  spirilla  come  into  contact  with  a 
leucocyte,  and  even  become  attached  by  one  end  to  its  surface  ;  yet,  how- 
ever active  the  movement,  one  never  finds  that  the  spirillum  succeeds  in 
piercing  the  surface  and  gaining  an  entrance.  If  it  be  suggested  that  this 
entry  may  take  place  in  consequence  of  the  force  of  active  growth  and  elon- 


2G2  SUSCEPTIBILITY  AND   IMMUNITY. 

Cation  of  bacilli,  then,  apart  from  the  fact  that  here  but  one  set  of  cases  is 
embraced,  it  can  be  determined  that  this  force  is  too  feeble — it  can  be  seen 
that,  during  the  active  growth  of  the  anthrax  organism  in  the  blood,  the 
elongating  chains  of  bacilli  curve  in  and  out  between  the  corpuscles,  but 
never  penetrate  the  cells. 

From  another  side  the  objection  has  been  formulated  that  in  many  cases 
the  organism  fleets  rid  of  its  invaders  without  the  aid  of  the  phagocytes. 
According  to  those  who  support  this  objection,  this  happens  in  the  anthrax 
of  pigeons  (CzaplewskiJ  and  of  refractory  rats  (Behring,  Franck),  in  symp- 
tomatic anthrax  of  various  refractory  animals  (Rogowicz),  and  in  the  septi- 
ca-mia  of  vaccinated  guinea  pigs,  due  to  the  Vibrio  Metschnikovi  (R.  Pfeif- 
fer).  A  reexamination  of  the  cases  here  adduced  has,  however,  shown 
that  in  each  a  very  considerable  phagocytosis  can  be  proved,  and  that  the 
negative  results  of  the  above  observers  have  been  due  to  insufficient  methods 
of  observation. 

While  accepting  that  the  phagocytes  do  truly  absorb  the  microorgan- 
isms, other  opponents  of  the  theory  have  urged  that  these  cells  are  only 
capable  of  including  microorganisms  already  killed  by  other  means,  and 
that  living  microbes  are  solely  to  be  found  within  the  cells  in  those  cases 
where  there  has  been  a  fatal  ending — in  tuberculosis,  mouse  septicaemia,  and 
so  on.  Against  this  may  be  brought  the  fact  determined  by  Lubarsch,  that 
the  phagocytes  of  several  animals,  refractory  to  anthrax,  take  up  living  ba- 
cilli that  have  been  injected,  with  greater  eagerness  than  they  include  those 
which  have  been  killed  before  injection.  But,  further,  this  objection  may 
be  disposed  of  by  direct  observation  of  bacteria  undergoing  development 
from  within  the  interior  of  phagocytes  after  the  latter  hayel>een  destroyed 
by  a  substance  which  is  at  tne  same  time  a  favorable  medium  for  bacterial 
growth — as,  for  instance,  beef  broth.  Such  observations  have  been  made 
upon  pigeons  rendered  immune  to  anthrax. 

During  the  last  year  or  two  great  stress  has  been  laid  upon  the  fact  that 
the  body  humors  themselves  possess  most  marked  bactericidal  properties, 
and,  in  fact,  against  the  theory  of  phagocytosis  has  been  brought  another, 
baned  upon  this  power  of  the  humors  to  destroy  the  microorganisms.  Ob- 
server after  observer  has  remarked  that  in  blood  plasma,  defibrinated  blood, 
blood  serum,  and  in  the  blood  as  a  whole,  in  the  removed  aqueous  humor 
and  other  fluids  and  exudations  of  the  body,  many  species  of  bacteria  perish 
after  a  longer  or  shorter  interval ;  and  forthwith  an  endeavor  has  been 
made  to  find  in  these  facts  some  elucidation  of  the  phenomena  of  immunity. 
Yet  the  more  deeply  one  examines  into  the  question  the  more  one  is  con- 
vinced that  no  relationship  exists  between  the  two.  Thus  it  happens  often 
that  tho  bactericidal  property  is  more  developed  in  susceptible  species  than 
in  refractory  ;  so,  with  regard  to  the  anthrax  bacilli,  in  the  very  sensitive 
rabbit  the  bactericidal  properties  of  the  humors  are  more  pronounced  than 
they  are  m  the  refractory  dog ;  and  Bearing  and  Nissen,  the  two  who  al- 
most simultaneously  first  drew  our  attention  to  these  phenomena,  in  their 
-• 


combined  research,  recently  published,  admit  that,  as  against  the  bacteria 
of  anthrax,  pneumonia,  and  diphtheria,  this  bactericidal  property  exists  to 

iiH-sani.-d.--n-..  m  the  juices  of  animals  of  the  same  species,  whether  they 
be  susceptible  or -have  been  rendered  immune.  Often,  again,  it  has  been 
determined  that  the  blood  removed  from  the  organism  has  a  greater  power 
of  destroying  bacteria  than  it  has  within  the  organism.  A  small  quantity 
of  Wood  withdrawn  from  the  body  will,  in  certain  instances,  kill  a  mass  of 
I. acilli  greater  than  that  which,  injected  into  the  circulation,  would  inevi- 
tably cause  death.  Evident  ly.  therefore,  in  this  bactericidal  influence  extra- 
raMUlar  phenomena  enact  an  imp,, riant  role  phenomena,  that  is  which 
naveno  connection  with  what  occurs  in  the  living  refractory  organism 

•'"»  another  point  ot  view  strong  ar-nments  have  been  directed  against 

this  theory  of  the  tissue  fluids.    It  has  been  shown,  especially  by  the  re- 

•cnesof  M.  Hall  km.-,  that  the  death  of  the  bacteria  transported  into  or- 

llui.ls  is  largely  due  to  the  sudden  change  of  medium,  and  that,  in 


SUSCEPTIBILITY    AND   IMMUNITY.  263 

passing  from  one  medium  to  another  by  successive  slight  modifications  in 
the  fluid  of  growth,  it  is  easy  to  make  bacteria  live  in  fluids  which,  when 
the  change  of  environment  has  been  abrupt,  swiftly  lead  to  their  destruc- 
tion. 

In  order  to  gain  an  idea  as  to  the  part  played  in  the  refractory  animal  by 
the  fluids  and  the  phagocytes  respectively,  the  endeavor  has  been  made  to 
separate  the  two  by  placing  under  the  skin  of  frogs  (which  are  naturally 
immune  to  anthrax)  minute  packets  formed  of  filter  paper  or  of  animal 
membrane,  and  containing  the  bacilli.  The  paper,  while  permitting  the 
passage  of  fluid,  wards  off  the  wandering  amoeboid  cells  for  a  certain  time. 
Shielded  in  this  way  from  the  phagocytes,  though  exposed  to  the  action  of 
the  juices,  the  bacilli  grow  well  and  produce  the  characteristic  felted  mass 
of  anthrax  filaments.  Baumgarten  has  not  been  able  to  confirm  this  experi- 
ment, but  Hueppe  and  Lubarsch  have  repeatedly  verified  it. 

But  it  is  not  even  necessary  to  take  these  precautions  in  order  to  assure 
one's  self  that  anthrax  spores  germinate  in  the  juices  of  refractory  animals. 
Recently,  for  instance,  M.  Trapeznikoff  has  found  that,  when  these  spores 
are  injected  into  the  dorsal  lymph  sac  of  the  frog,  they  constantly  tend  to 
develop  into  bacilli,  whose  further  growth  is  stopped  by  the  phagocytes, 
which  include  them,  along  with  such  spores  as  have  not  had  time  to  germi- 
nate. Eventually  the  bacilli  so  absorbed  are  digested  by  their  hosts,  while 
the  included  spores  remain  intact,  although  incapable  of  giving  birth  to 
bacilli  for  so  long  a  time  as  the  phagocytes  remain  alive.  And  I  might  ad- 
duce other  similar  cases.  Such  a  comparative  examination  proves  that  in 
the  living  body  the  bactericidal  property  resides  in  the  phagocytes  and  not 
in  the  fluids. 

Still,  it  may  be  urged  that  possibly  these  cells,  which  can  thus  devour  and 
destroy  the  living  microbes,  are  only  in  a  position  to  attack  bacteria  whose 
virulence  has  already  been  lessened  by  other  means  Were  this  so,  the  mi- 
crobes present  in  a  refractory  organism  should  behave,  not  like  parasites, 
but  as  simple,  inoffensive  saprophytes.  Hence  these  microbes — powerless 
to  produce  upon  a  refractory  soil  the  toxic  substances  which  render  them 
pathogenic  and  dangerous — should  easily  be  included  and  destroyed;  so 
that,  according  to  this  hypothesis,  which  "has  frequently  been  brought  for- 
ward, the  phagocytes  play  a  purely  secondary  and  dependent  part,  waiting 
until  the  microbes  are  weakened  before  they  seize  upon  them.  In  favor  of 
this  view  the  fact  has  been  cited  that  certain  microorganisms  cultivated  in 
the  blood,  or  serum,  of  vaccinated  animals  become  attenuated,  so  that  they 
no  longer  induce  a  fatal  disease.  The  Bacillus  anthracis  grown  in  the  blood 
of  vaccinated  sheep  no  longer  kills  rabbits,  and,  according  to  Roger,  the 
Streptococcus  erysipelatos  grown  in  the  blood  of  vaccinated  rabbits  only 
occasions  a  slight  and  passing  disturbance  in  susceptible  members  of  the 
same  species.  But  here  again  we  are  dealing  with  fluids  withdrawn  from 
the  body,  and  so  modified  in  various  ways.  Let  us  make  an  observation 
more  strictly  to  the  point.  Take,  for  instance,  a  rabbit  vaccinated  against 
anthrax  and  inoculate  it  with  anthrax  bacilli,  thus  allowing  these  to  exist 
directly  within  the  refractory  organism.  Such  bacilli  as  are  not  destroyed 
preserve  their  virulence  for  a  sufficiently  long  period,  and  it  is  possible  to 
kill  a  guinea-pig  with  a  drop  of  exudation,  taken  from  the  region  of  injection 
thirty  hours  after  subcutaneous  inoculation,  eight  days  after  inoculation 
into  the  anterior  chamber  of  the  eye.  A  sojourn  of  so  long  duration  within 
the  vaccinated  organism,  then,  has  not  deprived  the  microbes  of  their  viru- 
lence, although  twenty-four  hours  suffice  to  completely  attenuate  the 
bacilli  cultivated  in  the  removed  blood  of  vaccinated  sheep. 

Years  ago  it  was  established  in  M.  Pasteur's  laboratory  that  the  refrac- 
tory organism,  instead  of  being  an  unfavorable  soil  for  the  preservation  of 
virulence,  tends  the  rather  to  reinforce  this  property.  To  exalt  the  viru- 
lence of  an  attenuated  microorganism,  one  always  employs,  not  animals 
very  susceptible  to  the  specific  disease,  but  those  which  are  slightly  suscep- 
tible, or  it  may  be,  under  many  circumstances,  refractory.  In  this  manner 


204  SUSCEPTIBILITY   AND  IMMUNITY. 

the  most  active  anthrax  virus  has  usually  been  obtained  by  passage  through 
birds,  notably  fowls  ;  the  greatest  virulence  of  chicken  cholera  was  gained 
by  passage  through  the  vaccinated  cock ;  and  quite  recently  M.  Malm  has 
shown  that  passage  of  the  anthrax  bacillus  through  the  organisms  of  dogs, 
which  of  all  mammals  are  the  most  refractory  in  this  respect,  increases  its 
virulence  in  a  most  remarkable  manner,  so  that  the  general  law  may  be  laid 
down  that  an  organism  which  is  but  slightly  susceptible  or  is  refractory  is 
able  not  only  to  preserve,  but  even  to  exalt,  the  virulence  of  bacteria.  The 
principal  argument  in  favor  of  the  hypothesis  that  pathogenic  microor- 
ganisms become  simple  inoffensive  saprophytes  when  they  find  themselves 
in  a  refractory  region,  loses  therefore  its  raison  d'etre. 

M.  Bouchard,  in  his  objection  to  the  theory  of  phagocytosis,  may  be  re- 
garded as  introducing  but  a  modification  of  this  hypothesis.  He  holds  that 
pathogenic  bacteria  placed  under  favorable  conditions  give  rise  to  substances 
which  hinder  the  inflammatory  process,  and  that  only  when  these  inhibi- 
tory substances  are  inadequately  represented  do  the  cells  intervene.  When, 
therefore,  the  organism  rendered  refractory  by  vaccination  becomes  an  un- 
favorable soil  for  the  production  of  these  inhibitory  bodies,  the  bacteria  can 
no  longer  prevent  the  inflammatory  reaction  ;  free  emigration  of  the  leuco- 
cytes ensues,  these  cells  seize  upon  the  impotent  microbes  and  put  a  stop  to 
their  further  growth.  In  this  theory  the  part  played  by  the  phagocytes  is 
again  secondary,  depending  upon  a  dearth  of  anti-inflammatory  substance. 

If  the  theory  could  be  accepted  in  certain  cases,  it  is  nevertheless  inap- 
plicable as  a  general  rule.  In  all  those  affections  which  are  characterized  by 
the  absence  of  leucocytes  upon  the  field  of  battle  there  is  certainly  no  lack 
of  inflammation.  The  very  reverse  obtains.  In  anthrax  affecting  small 
mammals,  just  as  in  the  vibrionic  septicaemia  of  pigeons  and  guinea-pigs,  and 
other  analogous  diseases,  we  find  that  there  is  a  very  distinct  dilatation  of 
the  vessels,  accompanied  by  great  exudation  ;  the  inflammatory  reaction  is 
well  marked ;  nothing  is  wanting  save  the  determination  of  the  white  cor- 
puscles. Or,  employing  yet  further  that  affection  which  is,  as  it  were,  the 
touchstone  of  the  bacteriologist,  a  still  clearer  proof  of  our  contention  is  to 
be  gained  if  we  inoculate  a  rabbit  on  the  one  ear  with  a  small  quantity  of 
virulent,  on  the  other  with  a  like  quantity  of  attenuated,  anthrax  virus.  In 
the  course  of  a  few  hours  the  external  signs  of  inflammation  are  far  more 
conspicuous  in  the  former  ;  the  vessels  are  greatly  enlarged  and  there  is 
literally  a  huge  exudation  of  clear  serous  fluid  into  the  part ;  in  the  latter 
the  external  signs  are  less  prominent,  but  examination  of  the  seat  of  inocu- 
lation shows  it  to  be  packed  with  leucocytes.  Consequently,  the  phenome- 
non we  are  discussing  is  to  be  explained,  not  by  an  absence  of  the  inflamma- 
tory process,  but  much  more  satisfactorily  by  a  negative  chemiotaxis  of  the 
leucocytes,  which,  instead  of  being  attracted  by  the  bacterial  products,  are 
repelled  ;  where  the  animal  is  vaccinated  or  refractory  a  much  slighter  in- 
flammation is  sufficient  to  produce  an  abundant  emigration  of  the  leu- 
cocytes. 

Recently  Behring  has  brought  forward  another  view  which  would  ex- 
plain immunity  in  a  wholly  different  way.  According  to  him,  the  bac- 
teria can  live,  and  even  preserve  their  virulence,  in  the  refractory  organism, 
I Mit  the  toxines  excreted  by  them  now  undergo  a  modification  so  as  to  be 
rendered  completely  inoffensive  for  the  animal.  And  to  this  "toxicide 
property "  of  the  organism  is  to  be  attributed  the  essential  quality  of  the 
immune  state.  It  is  impossible  to  pronounce  upon  the  arguments  that  have 
led  up  to  this  theory,  for  as  vet  they  have  not  been  circumstantially  set 
forth ;  but  already  one  can  declare  that  such  a  theory  is  in  no  wise  applicable 
to  the  phenomena  of  immunity  in  general.  In  three  diseases  remarkable 
for  their  u  renounced  toxic  character — vibrionic  septicaemia,  pyocyanic  dis- 
MM,  a  nd  hog  cholera  affecting  the  rabbit- as  shown  by  the  experiments  of 
Charrm,  Gamalem,  and  Selander,  the  toxines  are  so  little  attacked  by  the  re- 
fractory organism  that  the  same  quant ity  of  those  substances  (freed  from 
bacteria)  suffices  to  kill  an  animal  very  susceptible  to  one  or  other  disease, 


SUSCEPTIBILITY   AND    IMMUNITY.  265 

and  an  animal  vaccinated  against  it  and  thus  completely  immune.  So,  too, 
non-fatal  doses  of  these  toxines  produce  in  animals  of  the  two  categories  the 
same  febrile  and  inflammatory  reactions.  The  proof  is  clear  that  there  is  no 
special  destruction  of  toxines  in  the  refractory  animal,  and  that  the  ' '  toxicide 
property,"  if  it  exists,  is  not  one  whit  more  developed  after  vaccination  than 
before.  Passing-  in  review  all  these  counter  theories,  we  see  that  each  of 
them  can  only  be  applied  to  a  certain  number  of  facts  ;  in  some  an  attenu- 
ating or  even  bactericidal  influence  of  the  juices  is  relied  upon,  in  others  an 
anti-inflammatory  action,  in  yet  others  a  toxicide  property.  Still  the  pha- 
gocytic  reaction  is  the  only  constant  in  all  those  cases  of  immunity  and 
recovery  that  have  as  yet  been  sufficiently  studied,  and  while  certain  of  the 
factors  mentioned  (the  attenuating  and  toxicide  properties)  do  not  in  the 
least  touch  upon  the  continued  existence  or  otherwise  of  the  microorganism, 
the  bactericidal  power  of  the  phagocyte  puts  an  end  to  the  parasite  itself,  and 
thus  at  a  given  moment  prevents  further  manifestation  of  its  virulence,  or 
preserves  the  animal  attacked  at  a  time  when  the  toxicide  properties  would  be 
found  wanting,  and  the  microbe  remaining  alive  would  consequently  gain 
the  upper  hand. 

But  while  thus  placing  before  you  the  important  part  played  by  the  pha- 
gocytes, I  do  not  wish  it  to  be  thought  that  these  cells  are  unaided  in  their 
contest  by  other  defensive  means  possessed  by  the  organism.  This  is  far 
from  being  my  view.  Thus,  in  the  febrile  reaction,  we  see  a  puissant  auxil- 
iary very  definitely  favoring  the  work  of  the  phagocytes.  This  febrile  re- 
action has  only  to  be  inhibited — as  was  done  by  M.  Pasteur  in  the  anthrax 
of  fowls — and  animals  naturally  refractory  to  the  affection  succumb  to  the 
ravages  of  the  bacilli.  It  is  not  possible  at  the  present  time  to  state  fully 
and  accurately  all  these  influences  which  are  associated  in  aiding  phago- 
cytic  action,  but  already  we  have  the  right  to  maintain  that,  in  the  prop- 
erty of  its  amoeboid  cells  to  include  and  to  destroy  microorganisms,  the 
animal  body  possesses  a  formidable  means  of  resistance  and  defence 
against  these  infectious  agents.1 

We  are  disposed  to  agree  with  Metschnikoff  in  his  final  conclu- 
sion,,as  above  stated  in  italics.  But  in  view  of  experimental  evi- 
dence, to  be  referred  to  later,  we  cannot  accept  the  so-called  Metsch- 
nikoff theory  as  a  sufficient  explanation  for  the  facts  relating  to 
natural  and  acquired  immunity  in  general,  and  must  regard  phago- 
cytosis simply  as  a  factor  which,  in  certain  infectious  diseases,  ap- 
pears to  play  an  important  part  in  enabling  immune  animals  to  resist 
invasion  by  pathogenic  bacteria. 

Going  back  to  the  demonstrated  fact  that  susceptible  animals  may 
be  made  immune  by  inoculating  them  with  the  toxic  products  pro- 
duced during  the  growth  of  certain  pathogenic  bacteria,  we  may 
suppose  either  that  immunity  results  from  the  continued  presence  of 
these  toxic  products  in  the  body  of  the  inoculated  animal,  or  from  a 
tolerance  acquired  at  the  time  of  the  inoculation  and  subsequently 
retained — by  transmission  from  cell  to  cell,  as  heretofore  suggested. 
Under  the  first  hypothesis — retention  theory — immunity  may  be  ex- 
plained as  due  to  a  continued  tolerance  on  the  part  of  the  cellular  ele- 
ments of  the  body  to  the  toxic  substances  introduced  and  retained ; 
or  to  the  effect  of  these  retained  toxic  products  in  destroying  the 
pathogenic  bacteria,  or  in  neutralizing  their  products  when  these  are 

1  From  the  British  Medical  Journal. 


SUSCEPTIBILITY   AND  IMMUNITY. 

subsequently  introduced  into  the  body  of  the  immune  animal.  We 
<3annot  understand  how  toxic  substances  introduced  in  the  first  in- 
stance can  neutralize  substances  of  the  same  kind  introduced  at  a 
later  date.  There  is  something  in  the  blood  of  the  rat  which,  accord- 
ing to  Behring,  neutralizes  the  toxic  substances  present  in  a  filtered 
culture  of  the  tetanus  bacillus ;  but  whatever  this  substance  may  be, 
it  is  evidently  different  from  the  toxic  substance  which  it  destroys, 
and  there  is  nothing  in  chemistry  to  justify  the  supposition  last 
made.  Is  it,  then,  by  destroying  the  pathogenic  microorganism 
that  these  inoculated  and  retained  toxic  products  preserve  the  animal 
from  future  infection  ?  Opposed  to  this  supposition  is  the  fact  that 
the  blood  of  an  animal  made  immune  in  this  way,  when  removed 
from  the  body,  does  not  prove  to  have  increased  germicidal  power  as 
compared  with  that  of  a  susceptible  animal  of  the  same  species. 
Again,  these  same  toxic  substances  in  cultures  of  the  anthrax  bacillus, 
the  tetanus  bacillus,  the  diphtheria  bacillus,  etc. ,  do  not  destroy  the 
pathogenic  germ  after  weeks  or  months  of  exposure.  And  when  we 
inoculate  a  susceptible  animal  with  a  virulent  culture  of  one  of  these 
microorganisms,  the  toxic  substances  present  do  not  prevent  the  rapid 
development  of  the  bacillus  ;  indeed,  instead  of  proving  a  germicide, 
they  favor  its  development,  which  is  more  abundant  and  rapid  than 
when  attenuated  cultures  containing  less  of  the  toxic  material  are 
used  for  the  inoculation.  In  view  of  these  facts  we  are  unable  to 
adopt  the  view  that  acquired  immunity  results  from  the  direct  action 
of  the  products  of  bacterial  growth,  introduced  and  retained  in  the 
body  of  the  immune  animal,  upon  the  pathogenic  microorganism 
when  subsequently  introduced  or  upon  its  toxic  products. 

But  there  is  another  explanation  which,  although  it  may  appear 
a  priori  to  be  quite  improbable,  has  the  support  of  recent  experimen- 
tal evidence.  This  is  the  supposition  that  some  substance  is  formed 
i  n  the  bod  i i  <>f  I  lie  immune  animal  which  neutralizes  the  toxic 
products  of  the  pathogenic  microorganism.  How  the  presence  of 
these  toxic  products  in  the  first  instance  brings  about  the  formation 
of  an  "antitoxin"  by  which  they  are  neutralized  is  still  a  mystery; 
but  that  such  a  substance  is  formed  appears  to  be  proved  by  the  ex- 
peri  ni«  nu  of  Ogata,  Behring  and  Kitasato,  Tizzoni  and  Cattani,  G. 
and  F.  Klemperer,  and  others. 

Ogata  and  Jasuhara,  in  a  series  of  experiments  made  in  the  Hy- 
gienic Institute  at  Tokio  (1890),  discovered  the  important  fact  that 
the  blood  of  an  animal  immune  against  anthrax  contains  some  sub- 
stance which  neutralizes  the  toxic  products  of  the  anthrax  bacillus. 
When  cultures  were  made  in  the  blood  of  dogs,  frogs,  or  of  white 
rats,  which  animals  have  a  natural  immunity  against  anthrax,  they 
were  found  not  to  kill  mice  inoculated  with  them.  Further  experi- 


SUSCEPTIBILITY   AND    IMMUNITY.  267 

ments  showed  that  mice  inoculated  with  virulent  anthrax  cultures 
did  not  succumb  to  anthrax  septicaemia  if  they  received  at  the  same 
time  a  subcutaneous  injection  of  a  small  quantity  of  the  blood  of  an 
immune  animal.  So  small  a  dose  as  one  drop  of  frog's  blood  or  one- 
half  drop  of  dog's  blood  proved  to  be  sufficient  to  protect  a  mouse 
from  the  fatal  effect  of  an  anthrax  inoculation.  And  the  protective 
inoculation  was  effective  when  made  as  long  as  seventy-two  hours 
before  or  five  hours  after  infection  with  an  anthrax  culture.  Fur- 
ther, it  was  found  that  mice  which  had  survived  anthrax  infection  as 
a  result  of  this  treatment  were  immune  at  a  later  date  (after  several 
weeks)  when  inoculated  with  a  virulent  culture  of  the  anthrax 
bacillus. 

Behring  and  Kitasato  have  obtained  similar  results  in  their  ex- 
periments upon  tetanus  and  diphtheria,  and  have  shown  that  the 
blood  of  an  immune  animal,  added  to  virulent  cultures  before  in- 
oculation into  susceptible  animals,  neutralizes  the  pathogenic  power 
of  these  cultures.  . 

They  have  shown  by  experiment  that  the  blood  of  a  rabbit  which 
has  an  acquired  immunity  against  tetanus,  mixed  with  the  virulent 
filtrate  from  a  culture  of  the  tetanus  bacillus,  neutralizes  its  toxic 
power.  One  cubic  centimetre  of  this  filtrate  was  mixed  with  five 
cubic  centimetres  of  serum  from  the  blood  of  an  immune  rabbit  and 
allowed  to  stand  for  twenty-four  hours  ;  0. 2  cubic  centimetre  of  this 
injected  into  a  mouse  was  without  effect,  while  0.0001  cubic  centi- 
metre of  the  filtrate  without  such  admixture  was  infallibly  fatal  to 
mice.  The  mice  inoculated  with  this  mixture  remained  immune  for 
forty  to  fifty  days,  after  which  they  gradually  lost  their  immunity. 
The  blood  or  serum  from  an  immune  rabbit,  when  preserved  in  a 
dark,  cool  place,  retained  its  power  of  neutralizing  the  tetanus  tox- 
albumin  for  about  a  week,  after  which  time  it  gradually  lost  this 
power.  The  blood  of  chickens,  which  have  a  natural  immunity 
against  tetanus,  was  found  not  to  have  a  similar  power.  Behring 
and  Kitasato  have  also  shown  that  the  serum  of  a  diphtheria-immune 
rabbit  destroys  the  potent  toxalbumin  in  diphtheria  cultures.  It 
does  not,  however,  possess  any  germicidal  power  against  the  diph- 
theria bacillus. 

Ogata,  in  1891,  reported  that  he  had  succeeded  in  isolating  from  the 
blood  of  dogs  and  of  chickens  a  substance  to  which  he  ascribes  the  nat- 
ural immunity  of  these  animals  from  certain  infectious  diseases,  and 
the  power  of  their  blood  to  protect  susceptible  animals  from  the  same 
diseases.  This  substance  is  soluble  in  water  and  in  glycerin,  but  in- 
soluble in  alcohol  or  ether,  by  which  it  is  precipitated  without  being 
destroyed.  Its  activity  is  neutralized  by  acids,  but  not  by  weak 
alkaline  solutions.  Ogata  supposes  the  substance  isolated  by  him  to 


SUSCEPTIBILITY   AND   IMMUNITY. 

be  the  active  agent  in  blood  serum  by  which  certain  pathogenic  bac- 
teria are  destroyed,  as  shown  by  the  experiments  of  Nuttall,  Buchner, 
and  others.  Hankin  had  previously  isolated  an  albuminoid  sub- 
stance from  the  spleen  and  blood  of  the  rat,  to  which  he  ascribed  the 
immunity  of  this  animal  from  anthrax.  This  substance,  according 
to  the  author  named,  is  a  globulin;  it  is  insoluble  in  alcohol  and  in 
distilled  water,  and  does  not  dialyze. 

Tizzoni  and  Cattani  ascribe  the  protection  of  animals  which  have 
acquired  an  immunity  against  tetanus  to  the  presence  of  an  albumi- 
nous substance  which  they  call  the  tetanus-antitoxin.  This  they 
have  isolated  from  the  blood  of  immune  animals.  They  arrive  at 
the  conclusion  that  it  is  a  globulin,  or  a  substance  which  is  carried 
down  with  the  globulin  precipitate,  and  that  it  is  different  from  the 
globulin,  above  referred  to,  obtained  by  Hankin  from  animals  im- 
mune against  anthrax. 

G.  and  F.  Klemperer,  in  1891,  published  an  important  memoir  in 
which  they  gave  an  account  of  their  researches  relating  to  the  ques- 
tion of  immunity,  etc.,  in  animals  subject  to  the  form  of  septicaemia 
produced  by  the  Micrococcus  pneumonia  crouposaB.  They  were  able 
to  produce  immunity  in  susceptible  animals  by  introducing  into  their 
bodies  filtered  cultures  of  this  micrococcus,  and  proved  by  experiment 
that  this  immunity  had  a  duration  of  at  least  six  months.  They 
arrived  at  the  conclusion  that  the  immunity  induced  by  injecting  fil- 
tered cultures  is  not  directly  due  to  the  toxic  substances  present  in 
these  cultures,  but  that  they  cause  the  production  in  the  tissues  of  an 
antitoxin  which  has  the  power  of  neutralizing  their  pathogenic 
action.  The  toxic  substance  present  in  cultures  of  the  "diplococcus 
of  pneumonia"  they  call  "  pneumotoxin" ;  the  substance  produced  in 
the  body  of  an  artificially  immune  animal,  by  which  this  pneumo- 
toxin is  destroyed  if  subsequently  introduced,  they  call  "  anti -pneumo- 
toxin." 

Emmerich,  in  a  communication  made  at  the  meeting  of  the  In- 
ternational Congress  for  Hygiene  and  Demography,  in  London,  re- 
ported results  which  correspond  with  those  of  G.  and  F.  Klemperer 
so  far  as  the  production  of  immunity  is  concerned,  and  also  gave  an 
account  of  experiments  made  by  Donissen  in  which  the  injection  of 
twenty  to  twenty-five  cubic  centimetres  of  blood  or  expressed  tissue 
juices,  filtered  through  porcelain,  from  an  immune  rabbit  into  an 
unprotected  rabbit,  subsequently  to  infection  with  a  bouillon  culture 
of  "diplococcus  pneumonia,"  prevented  the  development  of  fatal 
septicaemia.  Even  when  the  injection  was  made  twelve  to  fifteen 
hours  after  infection,  by  inhalation,  the  animal  recovered. 

Emmerich  and  Mastraum  had  previously  reported  similar  results 
in  experiments  made  upon  mice  with  the  Bacillus  erysipelatos  suis 


SUSCEPTIBILITY   AND    IMMUNITY.  269 

(rothlauf  bacillus).  White  mice  are  very  susceptible  to  the  patho- 
genic action  of  this  bacillus.  But  mice  which,  subsequently  to  in- 
fection, were  injected  with  the  expressed  and  filtered  tissue  juices  of 
an  immune  rabbit,  recovered,  while  the  control  animals  succumbed. 
According  to  Emmerich,  the  result  in  these  experiments  was  due  to 
a  destruction  of  the  pathogenic  bacilli  in  the  bodies  of  the  infected 
animals  ;  and  the  statement  is  made  that  at  the  end  of  eight  hours 
after  the  injection  of  the  expressed  tissue  juices  all  bacilli  in  the  body 
of  the  infected  animal  were  dead.  The  same  liquid  did  not,  however, 
kill  the  bacilli  when  added  to  cultures  external  to  the  body  of  an 
animal.  The  inference,  therefore,  seems  justified  that  the  result  de- 
pends, not  upon  a  substance  present  in  the  expressed  juices  of  an 
immune  animal,  but  upon  a  substance  formed  in  the  body  of  the 
animal  into  which  these  juices  are  injected. 

We  have,  however,  an  example  of  induced  immunity  in  which 
the  result  appears  to  depend  directly  upon  the  destruction  of  the 
pathogenic  microorganism  in  the  body  of  the  immune  animal.  In 
guinea-pigs  which  have  an  acquired  immunity  against  Vibrio  Metsch- 
nikovi  the  blood  serum  has  been  proved  to  possess  decided  germicidal 
power  for  this  "vibrio,"  whereas  it  multiplies  readily  in  the  blood 
serum  of  non-immune  guinea-pigs  (Behring  and  Nissen). 

There  is  experimental  evidence  that  animals  may  acquire  an  arti- 
ficial immunity  against  the  toxic  action  of  certain  toxalbumins  from 
other  sources  than  bacterial  cultures.  Thus  Sewell  (1887)  has  shown 
that  a  certain  degree  of  tolerance  to  the  action  of  rattlesnake  venom 
may  be  established  by  inoculating  susceptible  animals  with  small 
doses  of  the  "  hemialbumose "  to  which  it  owes  its  toxic  potency. 
These  results  have  been  confirmed  by  the  more  recent  experiments  Of 
Calmette  (1894)  and  of  Fraser  (1895).  In  his  paper  detailing  the 
results  of  his  experiments  the  first-named  author  says : 

' '  Animals  may  be  immunized  against  the  venom  of  serpents  either  by 
means  of  repeated  injections  of  doses  at  first  feeble  and  progressively  stronger, 
or  by  means  of  successive  injections  of  venom  mixed  with  certain  chemical 
substances,  among  which  I  mention  especially  chloride  of  gold  and  the  hypo- 
chlorites  of  lime  or  of  soda. 

"The  serum  of  animals  thus  treated  is  at  the  same  time  preventive,  anti- 
toxic, and  therapeutic,  exactly  as  is  that  of  animals  immunized  against 
diphtheria  or  tetanus. 

"If  we  inoculate  ascertain  number  of  rabbits,  under  the  skin  of  the 
thigh,  with  the  same  dose,  one  milligramme  of  cobra  venom  for  example, 
and  if  we  treat  all  of  these  animals  with  the  exception  of  some  for  control, 
by  subcutaneous  or  intraperitoneal  injections  of  the  serum  of  rabbits  im- 
munized against  four  milligrammes  of  the  same  venom,  all  of  the  control 
animals  not  treated  will  die  within  three  or  four  hours,  while  all  of  the 
animals  will  recover  which  receive  five  cubic  centimetres  of  the  therapeutic 
serum  within  an  hour  after  receiving  the  venom." 

In  this  connection  we  may  remark  that  there  is  some  evidence  to 


270  SUSCEPTIBILITY  AND  IMMUNITY. 

show  that  persons  who  are  repeatedly  stung  by  certain  poisonous  in- 
sects—mosquitoes, bees — acquire  a  greater  or  less  degree  of  immu- 
nity from  the  distressing  local  effects  of  their  stings. 

Ehrlich,  of  Berlin,  in  1891,  reported  his  success  in  establishing 
immunity  in  guinea-pigs  against  two  toxalbumins  of  vegetable 
origin:  one — ricin— from  the  castor-oil  bean  (Ricinus  communis), 
the  other— abrin— from  the  jequirity  bean.  The  toxic  potency 
of  ricin  is  somewhat  greater  than  that  of  abrin,  and  it  is  esti- 
mated by  Ehrlich  that  one  gramme  of  this  substance  would  suffice 
to  kill  one  and  a  half  millions  of  guinea-pigs.  When  injected  be- 
neath the  skin,  in  dilute  solution,  it  produces  intense  local  inflamma- 
tion, resulting  in  necrosis  of  the  tissues.  Mice  are  less  susceptible 
than  guinea-pigs  and  are  more  easily  made  immune.  This  is  most 
readily  effected  by  giving  them  small  and  gradually  increasing  doses 
with  their  food.  As  a  result  of  this  treatment  the  animal  resists 
subcutaneous  injections  of  two  hundred  to  four  hundred  times  the 
fatal  dose  for  animals  not  having  this  artificial  immunity.  The  fatal 
dose  of  abrin  is  about  double  that  of  ricin.  When  injected  into  mice 
in  the  proportion  of  one  cubic  centimetre  to  twenty  grammes  of  body 
weight  a  solution  of  one  part  in  one  hundred  thousand  of  water 
proved  to  be  a  fatal  dose.  The  local  effects  are  also  less  pronounced 
when  solutions  of  abrin  are  used  ;  they  consist  principally  in  an  ex- 
tensive induration  of  the  tissues  around  the  point  of  injection  and  a 
subsequent  falling  off  of  the  hair  over  this  indurated  area.  When 
introduced  into  the  conjunctival  sac,  however,  abrin  produces  a 
local  inflammation  in  smaller  amounts  than  ricin,  a  solution  of  1 : 800 
being  sufficient  to  cause  a  decided  but  temporary  conjunctivitis. 
Solutions  of  1 :  50  or  1 : 100  of  either  of  these  toxalbumins,  introduced 
into  the  eye  of  a  mouse,  give  rise  to  a  panophthalmitis  which  com- 
monly results  in  destruction  of  the  eye.  But  in  mice  which  have 
been  rendered  immune  by  feeding  them  for  several  weeks  with  food 
containing  one  of  these  toxalbumins,  no  reaction  follows  the  intro- 
duction into  the  eye  of  the  strongest  possible  solution,  or  of  a  paste 
made  by  adding  abrin  to  a  little  ten-per-cent  salt  solution.  Ehrlich 
gives  the  following  explanation  of  the  remarkable  degree  of  im- 
munity established  in  his  experiments  by  the  method  mentioned: 

"  All  of  these  phenomena  depend,  as  may  be  easily  shown,  upon 
the  fact  that  the  blood  contains  a  body — antiabrin — which  completely 
neutralizes  the  action  of  the  abrin,  probably  by  destroying  this  body." 

In  a  more  recent  paper  Ehrlich  has  given  an  account  of  subse- 
quent experiments  which  show  that  the  young  of  mice  which  have 
an  acquired  immunity  for  these  vegetable  toxalbumins  may  acquire 
immunity  from  the  ingestion  of  the  mother's  milk  ;  and  also  that 
immunity  against  tetanus  may  be  acquired  in  a  very  brief  time  by 


SUSCEPTIBILITY    AND   IMMUNITY.  271 

young  mice  through  their  mother's  milk.  In  his  tetanus  experi- 
ments Ehrlich  used  blood  serum  from  an  immune  horse  to  give  im- 
munity to  the  mother  mouse  when  her  young  were  already  seven- 
teen days  old.  Of  this  blood  serum  two  cubic  centimetres  were 
injected  at  a  time  on  two  successive  days.  The  day  after  the  first 
injection  one  of  the  sucklings  received  a  tetanus  inoculation  by 
means  of  a  splinter  of  wood  to  which  spores  were  attached.  The 
animal  remained  in  good  health,  while  a  much  larger  control  mouse 
inoculated  in  the  same  way  died  of  tetanus  at  the  end  of  twenty-six 
hours.  Other  sucklings,  inoculated  at  the  end  of  forty-eight  and  of 
seventy-two  hours  after  the  mother  had  received  the  injection  of 
blood  serum,  likewise  remained  in  good  health,  while  other  control 
mice  died. 

The  possibility  of  conferring  immunity  by  means  of  the  milk  of 
an  immune  animal  is  further  shown  by  the  experiments  of  Brieger 
and  Ehrlich  (1892).  A  female  goat  was  immunized  against  tetanus 
by  the  daily  injection  of  "  thymus-tetanus  bouillon."  The  dose  was 
gradually  increased  from  0.2  cubic  centimetre  to  10  cubic  centimetres. 
At  the  end  of  thirty-seven  days  a  mouse,  which  received  0.1  cubic 
centimetre  of  the  milk  of  this  goat  in  the  cavity  of  the  abdomen, 
proved  to  be  immune  against  tetanus.  Further  experiments  gave  a 
similar  result,  even  when  the  milk  of  the  goat  was  not  injected  into 
the  peritoneal  cavity  of  the  mouse  until  several  hours  after  inocu- 
lation with  a  virulent  culture  of  the  tetanus  bacillus. 

When  the  casein  was  separated  the  milk  retained  its  full  im- 
munizing activity,  and  by  concentration  in  vacuo  a  thick  milk 
was  obtained  which  had  a  very  high  immunization  value — 0.2  cubic 
centimetre  of  this  milk  protected  a  mouse  against  forty-eight  times 
the  lethal  dose  of  a  tetanus  culture. 

In  a  subsequent  communication  (1893)  Brieger  and  Ehrlich  de- 
scribe their  method  of  obtaining  the  antitoxin  of  tetanus  from  milk 
in  a  more  concentrated  form.  They  found  by  experiment  that  it  was 
precipitated  by  ammonium  sulphate  and  magnesium  sulphate.  From 
twenty-seven  to  thirty  per  cent  of  ammonium  sulphate  added  to  milk 
caused  a  precipitation  of  the  greater  part  of  the  antitoxin.  This  pre- 
cipitate was  dissolved  in  water,  dialyzed  in  running  water,  then 
filtered  and  evaporated  in  shallow  dishes  at  35°  C.  in  a  vacuum. 
One  litre  of  milk  from  an  immune  goat  gave  about  one  gramme  of  a 
transparent,  yellowish-white  precipitate,  which  contained  fourteen 
per  cent  of  ammonium  sulphate.  This  precipitate  had  from  four 
hundred  to  six  hundred  times  the  potency  of  the  milk  from  which 
it  was  obtained  in  neutralizing  the  tetanus  toxin. 

In  a  still  later  communication  (1893)  Brieger  and  Cohn  give  an 
improved  method  of  separating  the  antitoxin  from  the  precipitate 


272  SUSCEPTIBILITY  AND   IMMUNITY. 

thrown  down  with  ammonium  sulphate.  The  finely  pulverized  pre- 
cipitate is  shaken  up  with  pure  chloroform,  and  when  this  is  allowed 
to  stand  the  antitoxin  rises  to  the  surface  while  the  ammonium  salt 
sinks  to  the  bottom.  By  filling  the  vessel  to  the  margin  with  chloro- 
form, the  antitoxin  floating  on  the  surface  can  be  skimmed  off,  after 
which  it  quickly  dries.  By  this  method  the  considerable  loss  which 
occurred  in  the  dialyzer,  used  in  the  previously  described  method,  is 
avoided. 

A  most  interesting  question  presents  itself  in  connection  with  the 
discovery  of  the  antitoxins.  Does  the  animal  which  is  immune 
from  the  toxic  action  of  any  particular  toxalbumin  also  have  an  im- 
munity for  other  toxic  proteids  of  the  same  class?  The  experimental 
evidence  on  record  indicates  that  it  does  not.  In  Ehrlich's  experi- 
ments with  ricin  and  abrin  he  ascertained  that  an  animal  which  had 
been  made  immune  against  one  of  these  subtances  was  quite  as  sus- 
ceptible to  the  toxic  action  of  the  other  as  if  it  did  not  possess  this 
immunity,  i.e.,  the  antitoxin  of  ricin  does  not  destroy  abrin,  and 
vice  versa.  As  an  illustration  of  the  fact,  he  states  that  in  one  ex- 
periment a  rabbit  was  made  immune  for  ricin  to  such  an  extent  that 
the  introduction  into  its  eye  of  this  substance  in  powder  produced  no 
inflammatory  reaction ;  but  the  subsequent  introduction  of  a  solution 
of  abrin,  of  1  to  10,000,  caused  a  violent  inflammation. 

Evidently  these  facts  are  of  the  same  order  as  those  relating  to 
immunity  from  infectious  diseases,  and,  taken  in  connection  with  the 
experimental  data  previously  referred  to,  give  strong  support  to  the 
view  that  the  morbid  phenomena  in  all  diseases  of  this  class  are  due 
to  the  specific  toxic  action  of  substances  resembling  the  toxalbumins 
already  discovered  ;  and  that  acquired  immunity  from  any  one  of 
these  diseases  results  from  the  formation  of  an  antitoxin  in  the  body 
of  the  immune  animal. 

Hankin  calls  these  substances  produced  in  the  bodies  of  immune 
animals  "  defensive  proteids,"  and  proposes  to  classify  them  as  fol- 
lows :  First,  those  occurring  naturally  in  normal  animals,  which  he 
calls  sozins  ;  second,  those  occurring  in  animals  that  have  acquired 
an  artificial  immunity — these  he  calls  phylaxins.  Each  of  these 
classes  of  defensive  proteids  is  further  subdivided  into  those  which 
act  upon  the  pathogenic  microorganism  itself  and  those  which  act 
upon  its  toxic  products.  These  subclasses  are  distinguished  by  the 
prefixes  myco  and  t oxo  attached  to  the  class  name. 

In  accordance  with  this  classification  a  mycosozin  is  a  defensive 
proteid,  found  in  the  body  of  a  normal  animal,  which  has  the  power 
of  destroying  bacteria. 

A  toxosozin  is  a  defensive  proteid,  found  in  the  body  of  a  normal 


SUSCEPTIBILITY   AND    IMMUNITY.  273 

animal,  which  has  the  power  of  destroying  the  toxic  products  of  bac- 
terial growth. 

A  mycophylaxin  is  a  defensive  proteid  produced  in  the  body  of 
an  animal  which  has  an  acquired  immunity  for  a  given  infectious 
disease,  which  has  the  power  of  destroying  the  pathogenic  bacteria 
to  which  the  disease  is  due. 

A  toxophylaxin  is  a  defensive  proteid  produced  in  the  body  of 
an  animal  which  has  an  acquired  immunity  for  a  given  infectious 
disease,  which  has  the  power  of  destroying  the  toxic  products  of  the 
pathogenic  bacteria  to  which  the  disease  is  due. 

Buchner  had  previously  proposed  the  name  "  alexines  "  for  these 
defensive  proteids. 

The  importance  of  the  experimental  evidence  above  referred  to  in 
explaining  the  phenomena  of  natural  and  acquired  immunity  is  ap- 
parent. The  facts  stated  also  suggest  a  rational  explanation  of  re- 
covery from  an  attack  of  an  acute  infectious  disease.  But  the  idea 
that  during  such  an  attack  an  antidote  to  the  disease  poison  is  de- 
veloped in  the  tissues  is  yet  so  novel,  and  the  experimental  evidence 
in  support  of  this  view  is  of  such  recent  date,  that  it  would  be  pre- 
mature to  accept  this  explanation  as  applying  to  immunity  in  gene- 
ral. It  seems  difficult  to  believe  that  an  individual  who  has  passed 
through  attacks  of  measles,  mumps,  whooping  cough,  scarlet  fever, 
small-pox,  etc. ,  has  in  his  blood  or  tissues  a  store  of  the  antitoxine  of 
each  of  these  diseases,  formed  during  the  attack  and  retained  during 
the  remainder  of  his  life,  or  continuously  produced  so  long  as  the 
immunity  lasts.  Moreover,  in  those  diseases  to  which  the  experi- 
mental evidence  above  recorded  relates — diphtheria,  tetanus,  pneu- 
monia— as  they  occur  in  man,  no  lasting  immunity  has  been  shown 
to  result  from  a  single  attack,  and  in  this  regard  they  do  not  come 
into  the  same  class  with  the  eruptive  fevers  and  other  diseases  in 
which  a  single  attack  usually  protects  during  the  lifetime  of  the  in- 
dividual. 

In  those  instances  in  which  acquired  immunity  has  been  shown 
to  be  due  to  the  production  in  the  body  of  the  immune  animal  of  an 
antitoxin,  it  is  still  uncertain  whether  there  is  a  continuous  produc- 
tion of  the  protective  proteid,  or  whether  that  formed  during  the 
attack  remains  in  the  body  during  the  subsequent  immunity.  The 
latter  supposition  appears  at  first  thought  improbable  ;  but  when  we 
remember  that  the  protective  proteids  which  have  been  isolated  by 
Hankin  from  the  blood  and  spleen  of  rats,  and  by  Tizzoni  and  Cat- 
tani  from  the  blood  of  animals  made  immune  against  tetanus,  do 
not  dialyze,  it  does  not  seem  impossible  that  these  substances  might 
be  retained  indefinitely  within  the  blood-vessels.  On  the  other  hand, 
the  passage  of  the  tetanus  antitoxin  into  the  mother's  milk,  as 
18 


274  SUSCEPTIBILITY   AND   IMMUNITY. 

shown  by  Ehrlich's  experiments  upon  mice,  indicates  a  continuous 
supply,  otherwise  the  immunity  of  the  mother  would  soon  be  lost. 

The  writer  has  obtained  (May,  1892)  experimental  evidence  that 
the  blood  of  vaccinated,  and  consequently  immune,  calves  contains 
something  which  neutralizes  the  specific  virulence  of  vaccine  virus, 
both  bovine  and  humanized.  Four  drops  of  blood  serum  from  a  calf 
which  had  been  vaccinated  two  weeks  previously,  mixed  with  one 
drop  of  liquid  lymph  recently  collected  in  a  capillary  tube,  after  con- 
tact for  one  hour  was  used  to  vaccinate  a  calf ;  the  same  animal  was 
also  vaccinated  with  lymph,  preserved  on  three  quills,  which  was 
mixed  with  four  drops  of  serum  from  the  immune  calf  and  left  for 
one  hour.  The  result  of  these  vaccinations  was  entirely  negative, 
while  vaccinations  upon  the  same  calf  made  with  virus  from  the 
same  source,  and  mixed  with  the  same  amount  of  blood  serum  from 
a  non-immune  calf,  gave  a  completely  successful  and  typical  result, 

The  experimental  evidence  detailed  shows  that  in  certain  dis- 
eases acquired  immunity  depends  upon  the  formation  of  anti- 
toxins in  the  bodies  of  immune  animals-  As  secondary  fac- 
tors it  is  probable  that  tolerance  to  the  toxic  products  of  pathogenic 
bacteria  and  phagocytosis  have  considerable  importance,  but  it  is 
evident  that  the  principal  role  cannot  be  assigned  to  these  agencies. 

As  a  rule  the  antitoxins  have  no  bactericidal  action;  but  it  has 
been  shown  by  the  experiments  of  Gamaleia,  Pfeiffer,  and  others, 
that  in  animals  which  have  an  acquired  immunity  against  the  spiril- 
lum of  Asiatic  cholera  and  against  spirillum  Metschnikovi,  there  is  a 
decided  increase  in  the  bactericidal  power  of  the  blood  serum,  and 
that  immunity  probably  depends  upon  this  fact. 

The  researches  of  Metschnikoff  upon  hog  cholera,  of  Issaef  upon 
pneumonia,  and  of  Sanarelli  upon  typhoid  fever  indicate  that  the 
immunity  conferred  upon  susceptible  animals  by  protective  inocula- 
tions is  not  due  to  an  antitoxin  but  to  a  substance  present  in  the 
blood  of  immune  individuals  which  acts  directly  upon  the  pathogenic 
microorganism,  as  is  the  case  in  cholera-immune  animals.  The  ani- 
mals immunized  are  said  to  be  quite  as  sensitive  to  the  action  of  the 
bacterial  poisons  as  are  those  which  have  not  received  protective 
inoculations.  "Their  serum  does  not  protect  against  the  toxin,  but 
against  the  microbe"  (Roux). 


PLATE  IV. 

FIGS.  1,  2,  and  3. — Leucocytes  from  the  spleen  of  an  inoculated  monkey, 
containing  Spirillum  Obermeieri.  (Soudake witch.) 

Fias.  4  and  5. —Leucocytes  ("  macrophages  ")  from  a  preparation  of 
muscle  from  a  pigeon  which  succumbed  to  an  anthrax  inoculation.  In  Fig. 
4  the  bacilli  are  deeply  stained  ;  in  Fig.  5  they  are  pale.  (Metschnikoff.) 

FIG.  6. — Leucocyte  from  a  frog  seventy-two  hours  after  the  injection  of 
anthrax  spores.  (Trapeznikoff.) 

FIGS.  7  and  8. — Leucocytes  from  a  chicken  four  hours  after  the  injection 
of  anthrax  spores.  (Trapeznikoff.) 


Brt.  pp.  174- 71S. 


STEKNBERG'S  BACTERlOIflGT. 


P 1  at  e  TV. 


Fig. I. 


Fig. 2. 


3. 


Fig.  5. 


Fig  .6. 


Fio  7. 


PHAGOCYTES 


IV. 
PYOGEKEC   BACTERIA. 

THE    demonstration  made  by  Ogston,  Rosenbach,  Passet,  and 
others  that  micrococci  are  constantly  present  in  the  pus  of  acute 
abscesses,  led  to  the  inference  that  there  can  be  no  pus  formation  in 
the  absence  of  microorganisms  of  this  class.     But  it  is  now  well 
established,  by  the  experiments  of  Grawitz,   De  Bary,  Steinhaus, 
Scheurlen,  Kaufmann,  and  others,  that  this  inference  was  a  mis- 
taken one,  and  that  certain  chemical  substances  introduced  beneath 
the  skin  give  rise  to  pus  formation  quite  independently  of  bacteria. 
Among  the  substances  tested  which  have  given  a  positive  result  are 
nitrate  of  silver,  oil  of  turpentine,  strong  liquor  ammonias,  cada- 
verin,  etc.     The  demonstration  has  also  been  made  by  numerous  in- 
vestigators that  cultures  of  pus  cocci,  when  sterilized  by  heat,  still 
give  rise  to  pus  formation  when  injected  subcutaneously.     This  was 
first  established  by  Pasteur  in  1878,  who  found  that  sterilized  cul- 
tures of  his  "  microbe  generateur  du  pus  "  induced  suppuration  as 
well  as  cultures  containing  the  living  microbe.     This  fact  lias  since 
been  confirmed,  as  regards  the  pus  staphylococci  and  various  bacilli, 
by  a  number  of  bacteriologists.     Wyssokowitsch  produced  abscesses 
containing  sterile  pus  by  injecting  subcutaneously  agar  cultures  of 
the  anthrax  bacillus  sterilized  by  heat.     Buchner  obtained  similar 
results  in  a  series  of  forty  experiments  from  the  injection  of  steril- 
ized cultures  of  Friedlander's  bacillus  ("  pneumococcus  ")>  and  has 
shown  that  the  pus-forming  property  belongs  to  the  bacterial  cells 
and  not  to  a  soluble  chemical  substance  produced  by  them.     When 
cultures  were  filtered  by  means  of  a  Chamberlain  filter  the  clear 
fluid  which  passed  through  the  porous  porcelain  was  without  effect, 
while  the  dead  bacteria  retained  by  the  filter  produced  aseptic  pus 
infiltration  in  the  subcutaneous    tissues  within  forty-eight    hours 
after  having  been  injected.     Subsequent  experiments  gave  similar 
results  with  seventeen  different  species  tested,  including  Staphylo- 
coccus  pyogenes  aureus,  Staphylococcus  cereus  flavus,  Sarcina  auran- 
tiaca,   Bacillus    prodigiosus,   Bacillus   Fitzianus,   Bacillus    subtilis, 
Bacillus  coli  communis,  Bacillus  acidi  lactici,  etc.     From  the  experi- 


276  PYOGENIC  BACTERIA. 

ments  made  to  determine  the  exact  cause  of  pus  formation  following 
the  injection  of  sterilized  cultures  Buchner  arrives  at  the  conclusion 
that  it  is  due  to  the  albuminous  contents  of  the  bacterial  cells. 

While  it  is  demonstrated  that  a  large  number  of  microorganisms, 
either  living  or  in  sterilized  cultures,  may  give  rise  to  the  formation 

*•  "fr^of  pus,  the  extended  researches  of  Rosenbach,  Passet,  and  other 
bacteriologists  show  that  few  species  are  usually  concerned  in  the 
formation  of  acute  abscesses,  furuncles,  etc.,  in  man.  Of  these  the 
two  most  important,  by  reason  of  their  frequent  occurrence  and  path- 
ogenic power,  are  Staphylococcus  pyogenes  aureus  and  Strepto- 
coccus pyogenes ;  next  to  these  comes  Staphylococcus  pyogenes 
albus,  and  the  following  species  are  occasionally  found  :  Staphylo- 
coccus pyogenes  citreus,  Staphylococcus  cereus  flavus,  Staphylococcus 
cereus  albus,  Micrococcus  tenuis,  Bacillus  pyogenes  f  cetidus,  Micro- 
coccus  tetragenus,  Micrococcus  pneumonias  crouposae.  Two  or  more 
species  are  often  found  in  the  same  abscess  ;  thus  Passet,  in  thirty- 
three  cases  of  acute  abscess,  found  Staphylococcus  aureus  and  albus 
associated  in  eleven,  albus  alone  in  four,  albus  and  citreus  in  two, 
Streptococcus  pyogenes  alone  in  eight,  albus  and  streptococcus  in 
one,  and  albus,  citreus,  and  streptococcus  in  one.  Hoff a  found,  in 
twenty-two  cases  of  inguinal  bubo,  aureus  in  ten,  albus  in  nine,  and 
citreus  in  three.  Bumm,  in  ten  cases  of  puerperal  mastitis,  found 
aureus  in  seven  and  Streptococcus  pyogenes  in  three.  Rosenbach 
found  staphylococci  alone  sixteen  times,  Streptococcus  pyogenes  alone 
fifteen  times,  staphylococci  and  streptococci  associated  five  times, 
and  Micrococcus  tenuis  three  times  in  thirty-nine  acute  abscesses  and 
phlegmons  examined  by  him. 

.  ,oj  Robb  and  Ghrisky  have  shown  that  under  the  most  rigid  antisep- 
^  tic  treatment  microorganisms  are  constantly  found  attached  to  su- 
tures when  these  are  removed  from  wounds  made  by  the  surgeon, 
and  that  a  skin  abscess  frequently  results  from  the  presence  of  the 
most  common  of  these  microorganisms — Staphylococcus  epidermidis 
albus. 

The  authors  named  state  their  conclusions  as  follows  : 

*'  A  wound,  at  some  time  of  its  existence,  always  contains  organisms. 
They  occur  either  on  the  stitches  or  in  the  secretions. 

44  The  number  of  bacteria  is  influenced  by  the  constricting  action  of  the 
ligatures  or  drainage  tube,  or  anything  interfering  with  the  circulation  of 
the^tissues. 

4 'The  virulence  of  the  organisms  present  will  influence  the  progress  of 
the  wound. 

"The  bpdv  temperature  is  invariably  elevated  if  the  bacteria  are  viru- 
lent; and,  indeed,  in  cases  where  many  of  the  less  virulent  organisms  are 
found,  almost  without  exception  there  is  some  rise  of  temperature." 

The  organism  most  frequently  found — Staphylococcus  epidermi- 


PYOGENIC   BACTERIA.  277 

dis  albus — has  but  slight  virulence.  Out  of  forty-five  cases  in  which 
a  bacteriological  examination  was  made  this  micrococcus  was  ob- 
tained in  pure  cultures  in  thirty -three  ;  in  five  cases  it  was  associated 
with  Staphylococcus  pyogenes  aureus,  in  one  case  with  Streptococ- 
cus pyogenes,  in  three  cases  Streptococcus  pyogenes  was  obtained 
alone. 

In  abscesses  resulting  from  inflammation  of  the  middle  ear  the 
micrococcus  commonly  known  under  the  name  of  "  diplococcus 
pneumonias  " — Micrococcus  pneumoniae  crouposae — has  been  obtained 
in  pure  cultures  in  a  considerable  number  of  cases  when  the  pus  has 
been  examined  immediately  after  paracentesis  of  the  tympanic  mem- 
brane. We  shall  not,  however,  describe  this  among  the  pyogenic 
bacteria,  but  will  give  an  account  of  it  in  the  following  section  (Bac- 
teria in  Croupous  Pneumonia,  etc.).  Bacillus  pyocyaneus,  which  is 
described  by  some  authors  among  the  pyogenic  bacteria,  is  found 
only  in  the  pus  of  open  wounds,  where  its  presence  is  evidently  acci- 
dental. We  shall  describe  it  among  the  chromogenic  saprophytes. 

1.  STAPHYLOCOCCUS  PYOGENES  AUREUS. 

Synonym. — Micrococcus  of  infectious  osteomyelitis  (Becker). 

Observed  by  Ogston  (1881)  in  the  pus  of  acute  abscesses,  but  not 
differentiated  from  the  associated  staphylococci  and  the  streptococ- 
cus of  pus.  Obtained  by  Becker  from  the  pus  of  osteomyelitis  (1883). 
Isolated  from  the  pus  of  acute  abscesses  and  accurately  described  by 
Rosenbach  (1884)  and  by  Passet  (1885). 

The  Staphylococcus  pyogenes  aureus  is  a  facultative  parasite,  and  / 
is  the  most  common  pyogenic  micrococcus  found  in  suppurative  pro- 
cesses generally.  But  it  is  also  a  common  and  widely  distributed 
saprophyte,  which  finds  the  conditions  necessary  for  its  existence  on 
the  external  surface  of  the  human  body  and  of  moist  mucous  mem- 
branes. This  is  shown  by  the  researches  of  numerous  bacteriolo- 
gists. Thus  Ullmann  found  it  upon  the  skin  and  in  the  secretions  of 
the  mouth  of  healthy  persons,  and  also  in  the  dust  of  occupied  apart- 
ments, in  water,  etc.;  Bockhart  obtained  it  in  cultures  from  the 
surface  of  the  body  and  from  the  dirt  beneath  the  finger  nails  of 
healthy  persons  ;  Biondi,  Vignal,  and  others  in  the  salivary  secre- 
tions ;  B.  Frankel  in  mucus  from  the  pharynx ;  Von  Besser  and 
Wright  in  nasal  mucus  ;  Escherich  in  the  alvine  discharges  of 
healthy  infants  ;  C.  Frankel  in  the  air  ;  and  Liibbert  in  the  soil.  Its 
presence  in  the  air,  in  water,  or  in  the  soil  is,  however,  quite  excep- 
tional, and  is  probably  to  be  considered  the  result  of  accident,  its 
normal  habitat  as  a  saprophyte  appearing  to  be  rather  upon  the  sur- 
face  of  the  b9dy  and  of  mucous  membranes. 

v.o*^  ^         <N~VA~-<^ 

U&WNA* 


278  PYOGENIC  BACTERIA. 

Morphology.—  Spherical  cells  having  a  diameter  of  0.7  /i  (Hade* 
lich)  to  0.9  /i  (0.87  fit  Passet),  solitary,  in  pairs,  or  in  irregular 
groups,  occasionally  in  chains  of  three  or  four  elements  or  in  groups 
of  four.  The  dimensions  vary  somewhat  in  dif- 
ferent culture  media,  being  larger  in  a  favorable 
than  in  an  unfavorable  medium.  The  individual 
cells,  as  pointed  out  by  Hadelich,  consist  of  two 
hemispherical  portions  separated  from  each  other 
FIO.  79.-staphyiococ-  by  a  very  narrow  cleft,  which  is  not  visible  when 
tom^a^towiT^T'  the  ceUs  are  deePlv  stained,  but  may  be  demon- 
R^nbach.  Y  strated,  with  a  high  power,  by  staining  for  a  short 

time  (two  minutes  or  less)  in  a  solution  of  f  uchsin  in  aniline  water. 

This  micrococcus  stains  quickly  in  aqueous  solutions  of  the  basic 
aniline  colors,  and  may  also  be  stained  with  acid  carmine  and  haema- 
toxylin.  It  is  not  decolorized  by  iodine  solution  when  stained  with 
methyl  violet — Gram's  method. 

Biological  Characters.—  Staphylococcus  pyogenes  aureus  grows 
either  in  the  presence  or  absence  of  oxygen,  and  is  consequently  a 
facultative  anaerobic.  It  multiplies  rapidly  at  a  temperature  of  18° 
to  20°  C.  in  milk,  flesh  infusions,  and  various  other  liquid  media, 
and  in  nutrient  gelatin  or  agar.  It  liquefies  gelatin,  and  in  stick 
cultures  liquefaction  occurs  all  along  the  line  of  puncture,  forming  a 
pouch  which  is  largest  above  and  at  the  end  of  three  or  four  days  has 
extended  to  the  full  capacity  of  the  test  tube  at  the  surface.  The 
liquefied  gelatin  in  this  pouch  is  at  first  opaque  from  the  presence  of 
little  agglomerations  of  micrococci  in  suspension,  but  after  a  time 
these  are  deposited  and  the  gelatin  becomes  transparent.  During 
the  period  of  active  growth  the  cocci  accumulate  near  the  surface  of 
the  gelatin,  and,  in  contact  with  the  air,  the  characteristic  golden-yel- 
low pigment  is  produced.  By  the  subsidence  of  the  colored  masses 
of  cocci  from  this  superficial  stratum  a  yellow  deposit  is  gradually 
formed  at  the  bottom  of  the  pouch  of  liquefied  gelatin  (Fig.  80).  This 
pigment,  which  is  the  principal  character  distinguishing  the  micro- 
coccus  under  consideration  from  certain  other  liquefying  staphylo- 
cocci,  is  only  formed  in  the  presence  of  oxygen.  Upon  the  surface 

<  »f  nutrient  agar  development  occurs  in  the  form  of  a  moist,  shining 
layer,  with  more  or  less  wavy  outlines,  having  at  first  a  pale-yellow 
color,  which  soon  deepens  to  an  orange-  or  golden-yellow.     The  col- 

<  mies  which  develop  upon  agar  plates  are  spherical  and  opaque,  and 
usually  acquire  the  golden-yellow  color  within  a  few  days.     Colonies 
on  gelatin  plates  or  in  Esmarch  roll  tubes  first  appear  as  small  white 
dots,  which  later  are  more  or  less  granular  in  appearance  and  present 
the  yellow  color,  especially  towards  the  centre  ;  but,  owing  to  the 
extensive  liquefaction  of  the  gelatin  caused  by  them,  their  develop- 


PYOGENIC   BACTERIA. 


279 


ment  can  only  be  followed  for  two  or  three  days.  Upon  potato,  at  a 
temperature  of  35°  to  37°  C.,  a  rather  thick,  moist  layer  of  consider- 
able extent  forms  at  the  end  of  twenty-four  to  forty-eight  hours ; 
this  is  also  at  first  of  a  pale-yellow,  and  later 
of  an  orange-yellow  color.  The  temperature 
mentioned  is  most  favorable  for  the  rapid 
development  of  this  micrococcus,  although 
multiplication  may  occur  at  a  comparatively 
low  temperature  and  is  tolerably  abundant  at 
the  ordinary  room  temperature. 

Cultures  of  the  "golden  Staphylococcus/' 
and  especially  those  upon  potato,  give  off  a 
peculiar  odor  which  resembles  that  of  sour 
paste.  When  cultivated  in  milk  it  gives  rise 
to  the  formation  of  lactic  and  butyric  acids 
and  to  coagulation  of  the  casein.  No  poison- 
ous ptomaines  or  toxalbumins  have  been  iso- 
lated from  cultures  of  this  micrococcus,  but, 
like  other  liquefying  bacteria,  it  forms  a  sol- 
uble peptonizing  ferment,  by  which  gelatin 
may  be  liquefied  independently  of  the  living 
microorganism.  While  the  Staphylococcus 

aureus  gives  rise  to  the  production  of  acids —  Fia  go.-Geiatm  culture  of 
principally  lactic  acid — in  media  containing  Staphylococcus  pyogenes  aureus 
glucose  or  lactose,  it  has  also  been  shown  by  (Baum^arten)- 
Brieger  that  ammonia  is  one  of  the  products  of  its  vital  activity. 
Unlike  some  other  pathogenic  bacteria,  it  is  able  to  grow  in  a  medium 
having  a  distinctly  acid  reaction.  A  non-poisonous  basic  substance 
has  been  isolated  by  Brieger  from  old  cultures  in  meat  infusion  which 
differs  from  any  of  the  ptomaines  obtained  by  him  from  other  sources. 

The  thermal  death-point  of  this  micrococcus,  in  recent  cultures  in 
flesh-peptone-gelatin,  as  determined  by  the  writer,  is  between  56°  and 
58°  C.,  the  time  of  exposure  being  ten  minutes.  When  in  a  desic- 
cated condition  a  much  higher  temperature  is  required — 90°  to  100°  C. 
— for  its  destruction  ;  and  it  retains  its  vitality  for  more  than  ten 
days  when  dried  upon  a  cover  glass  (Passet).  It  retains  its  vitality 
for  a  long  time  in  cultures  in  nutrient  gelatin  or  agar,  and  may  grow 
when  transplanted  from  such  cultures  even  at  the  end  of  a  year. 

Very  numerous  experiments  have  been  made  to  determine  the 
proportion  of  various  chemical  agents  required  to  destroy  the  vitality 
or  to  restrain  the  growth  of  this  important  pyogenic  micrococcus. 
The  extended  researches  of  Liibbert  (1886)  with  reference  to  the 
antiseptic  power  of  agents  added  to  a  suitable  culture  medium — nu- 
trient gelatin — gave  the  following  results  :  Development  was  pre- 


PYOGENIC   BACTERIA. 

vented  by  the  agents  named  in  the  proportion  given  :  Nitric  acid, 
1  :  797  ;  phosphoric  acid,  1  :  750  ;  boracic  acid,  1  :  327  ;  oxalic  acid, 
1  : 433  ;  acetic  acid,  1  :  720  ;  citric  acid,  1  :  433  ;  lactic  acid,  1  : 350  ; 
benzoic  acid,  1  : 400 ;  salicylic  acid,  1  : 655  ;  iodine  dissolved  with 
potassium  iodide,  1:1,100;  arsenite  of  potash,  1:733;  mercuric 
chloride,  1  :  81,400  ;  chloral  hydrate,  1 : 133  ;  carbolic  acid,  1  :  814  ; 
thymol,  1  : 11,000  ;  resorcin,  1  : 122  ;  hydrochinon,  1  :  353  ;  kairin, 
1  : 407  ;  antipyrin,  1  :  26  ;  muriate  of  quinine,  1  : 550  ;  muriate  of 
morphia,  1  :  60.  For  the  destruction  of  vitality  very  much  larger 
amounts  are  required.  In  Bolton's  experiments  (1887)  a  one-per-cent 
solution  of  carbolic  acid  was  successful  after  two  hours'  exposure, 
but  two  per  cent  failed  to  completely  destroy  vitality  in  the  same 
time  ;  one  per  cent  of  sulphate  of  copper  was  also  successful,  and  but 
a  single  colony  developed  after  exposure  to  a  solution  of  1  : 200.  In 
the  experiments  of  Gartner  and  Plagge  the  Staphylococcus  aureusin 
bouillon  cultures  is  said  to  have  been  killed  in  a  few  seconds  (eight) 
by  a  solution  of  mercuric  chloride  of  the  proportion  of  1  : 1,000  ;  Behr- 
ing  found  it  was  killed  by  the  acid  sublimate  solution  of  La  Place, 
in  the  proportion  of  1  : 1,000,  in  ten  minutes ;  Tarnier  and  Vignal 
found  that  a  solution  of  1  : 1,000  was  successful  in  two  minutes. 
Abbott  (1891)  has  shown  that  in  the  same  culture  there  may  be  a 
considerable  difference  in  the  resisting  power  of  the  cocci,  and  that 
while  frequently  all  are  destroyed  in  five  minutes  by  a  1  : 1,000  solu- 
tion, it  occurs  quite  as  frequently  that  some  may  survive  after  an  ex- 
posure of  ten,  twenty,  and  even  thirty  minutes. 

Pathogenesis. — Subcutaneous  inoculation  with  a  small  quantity 
of  a  culture  of  Staphylococcus  pyogenes  aureus  is  without  result  in 
rabbits,  guinea-pigs,  or  mice,  but  when  a  considerable  quantity  is 
injected  beneath  the  skin  of  a  rabbit  or  a  guinea-pig  an  abscess  is 
produced,  which  usually  results  in  recovery,  but  may  give  rise  to 
general  infection  and  the  death  of  the  animal.  Injection  into  a 
vein  or  into  the  cavity  of  the  abdomen  in  the  animals  mentioned 
usually  induces  a  fatal  result  within  a  few  days.  The  most  charac- 
teristic pathological  changes  are  found  in  the  kidneys,  which  con- 
tain numerous  small  collections  of  pus  and  under  the  microscope 
present  the  appearances  resulting  from  embolic  nephritis.  Many  of 
the  capillaries  and  some  of  the  smaller  arteries  of  the  cortex  are 
plugged  up  with  thrombi  consisting  of  micrococci.  Metastatic  ab- 
scesses may  also  be  found  in  the  joints  and  muscles.  The  micro- 
cocci  may  be  recovered  in  pure  cultures  from  the  blood  and  the 
various  organs  ;  but  they  are  not  numerous  in  the  blood,  and  a  sim- 
ple microscopical  examination  will  often  fail  to  demonstrate  their 
presence. 

Animals  frequently  survive  the  injection  of  a  small  quantity  of 


PYOGENIC   BACTERIA. 


281 


a  pure  culture  macje  directly  into  the  circulation,  and  there  is  evi- 
dence that  the  pathogenic  potency  of  this  micrococcus  may  vary 
considerably  as  a  result  of  conditions  relating  to  its  origin  and  culti- 
vation in  the  animal  body  or  in  artificial  media.  When  injected  in 
considerable  quantities  it  may  be  obtained  in  cultures  from  the 
urine,  but  not  sooner  than  six  or  eight  hours  after  the  injection,  and 
not  until  the  formation  of  purulent  foci  in  the  kidneys  has  already 
occurred  (Wyssokowitsch). 

The  pyogenic  properties  of  this  micrococcus  have  been  demon-  ^ 
strated  upon  man  by  the  experiments  of  G-arre,  of  Bockhart,  and  of 
Bumm.     The  first-named  observer  inoculated  a  small  wound  at  the 
edge  of  one  of  his  finger  nails  with  a  minute  quantity  of  a  pure  cul- 
ture, and  a  subepidermal,  purulent  inflammation  extending  around 


FIG.  81.— Vertical  section  through  a  subcutaneous  abscess  caused  by  inoculation  with  staphylo- 
cocci,  in  the  rabbit,  forty-eight  hours  after  infection;  margin  towards  the  normal  tissue,  x  950. 
(Baumgarten.) 

the  margin  of  the  nail  resulted  from  the  inoculation.  Staphylococ- 
cus  aureus  was  recovered  in  cultures  from  the  pus  thus  formed.  A 
more  extensive  and  extremely  satisfactory  experiment  was  subse- 
quently made  by  Garre,  who  applied  a  considerable  quantity  of  a 
pure  culture  obtained  from  the  above-mentioned  source — third  gene' 
ration — to  the  uninjured  skin  of  his  left  forearm.  At  the  end  of 
four  days  a  large  carbuncle,  surrounded  by  isolated  furuncles,  de- 
veloped at  the  point  where  the  culture  had  been  applied.  This  ran 
the  usual  course,  and  it  was  several  weeks  before  it  had  completely 
healed.  No  less  than  seventeen  scars  remained  to  give  evidence  of 
the  success  of  the  experiment. 

In  Bockhart's  experiments  a  similar  but  milder  result  was  ob- 
tained,  the  conditions  having  been  somewhat  different.     A  small 


282  PYOGENIC   BACTERIA. 

quantity  of  an  agar  culture  was  suspended  in  O.^-per-cent  salt  solu 
tion,  and  this  was  rubbed  upon  the  uninjured  skin  of  the  left  fore- 
arm. By  gentle  scratching  with  a  disinfected  finger  nail  the  epithe- 
lium was  removed  in  places  over  the  area  to  which  the  micrococcus 
had  been  applied.  As  a  result  of  this  procedure  numerous  impe- 
tigo pustules  and  occasionally  a  genuine  furuncle  developed.  Por- 
tions of  the  skin  containing  the  smaller  pustules  were  excised  and 
examined  microscopically.  As  a  result  of  this  examination  Bock- 
hart  concluded  that  the  cocci  penetrate  by  way  of  the  hair  follicles, 
the  sebaceous  and  sudoriparous  glands,  or,  where  the  epidermis  had 
been  removed  by  scratching,  directly  to  the  deeper  layers  of  the  skin. 

In  Bumm's  experiments,  made  upon  himself  and  several  other 
persons,  Staphylococcus  aureus  suspended  in  sterilized  salt  solution 
was  injected  beneath  the  skin.  An  abscess  resulted  in  every  case. 

The  very  extended  researches  made  by  bacteriologists  during  the 
past  five  or  six  years  show  that  the  golden  staphylococcus  is  the 
most  common  pyogenic  microorganism.  Its  presence  has  been  de- 
monstrated not  only  in  furuncles  and  carbuncles,  but  also  in  various 
pustular  affections  of  the  skin  and  mucous  membranes — impetigo, 
sycosis,  phlyctenular  conjunctivitis  ;  in  purulent  conjunctivitis  and 
inflammation  of  the  lacrymal  sac  ;  in  acute  abscesses  formed  in  the 
lymphatic  glands,  the  parotid  gland,  the  tonsils,  the  mammae,  etc. ; 
in  metastatic  abscesses  and  purulent  collections  in  the  joints  ;  in  em- 
pyema  ;•  in  infectious  osteomyelitis  ;  and  in  ulcerative  endocarditis. 
The  evidence  relating  to  its  presence  and  etiological  import  in  the 
last-mentioned  affections  demands  special  consideration. 

Infectious  osteomyelitis  appears  from  the  researches  of  Becker, 
Rosenbach,  Krause,  Passet,  and  others,  to  be  usually  due  to  the  pre- 
sence of  Staphylococcus  aureus,  although  Kraske  has  shown  that  in 
certain  cases  this  is  associated  with  other  microorganisms.  Becker, 
who  obtained  this  micrococcus  from  the  pus  of  osteomyelitis  in  1883, 
was  the  first  to  show  by  experiment  that  the  same  affection  might  be 
induced  in  rabbits  by  injecting  cultures  of  the  micrococcus  into  the 
circulation,  after  having  crushed  or  fractured  a  bone  in  one  of  its 
legs.  The  animal  usually  died  in  from  twelve  to  fourteen  days  and 
presented  the  usual  appearances  of  osteomyelitis  at  the  fractured 
point.  The  abundant  yellowish-white  pus  contained  the  golden 
staphylococcus  which  was  described  by  Becker,  and  subsequently 
known  in  the  bacteriological  laboratories  of  Germany  as  the  "  mi- 
crococcus of  infectious  osteomyelitis."  Becker's  experimental  re- 
sults have  been  confirmed  by  Krause  and  Rosenbach;  and  Rodet,  by 
injecting  smaller  quantities  of  a  culture  into  the  circulation,  has  suc- 
ceeded in  producing  an  osteomyelitis  without  previous  injury  to  the 
bone. 


PYOGENIC   BACTERIA.  285 

Ulcerative  endocarditis  has  been  shown  by  the  researches  of 
numerous  bacteriologists  to  be  occasionally  accompanied  by  a  mycotic 
invasion  of  the  affected  tissues  by  the  golden  staphylococcus ;  in 
other  cases  Streptococcus  pyogenes  is  present.  The  researches  of 
Weichselbaum,  and  of  E.  Frankel  and  Sanger,  also  show  that  it  is 
present  in  a  certain  proportion  of  the  cases,  at  least,  of  endocarditis 
verrucosa,  although  in  smaller  numbers.  That  the  diseased  condi- 
tion of  the  cardiac  valves  in  ulcerative  endocarditis  is  due  to  mycotic 
invasion  is  now  generally  admitted  and  is  supported  by  experimental 
evidence.  Rosenbach  first  (1873)  produced  an  endocarditis  in  lower 
animals  by  mechanical  injury  to  the  cardiac  valves,  effected  by  in- 
troducing a  sound  through  the  aorta.  Following  his  method,  Wys- 
sokowitsch  (1885),  after  injuring  the  cardiac  valves  in  rabbits,  in- 
jected into  the  circulation  pure  cultures  of  various  bacteria.  He 
obtained  positive  results  with  Staphylococcus  aureus  and  Strepto- 
coccus pyogenes  only.  When  these  micrococci  were  injected  into 
the  trachea  or  subcutaneously  the  result  was  negative,  as  was  the 
case  when  very  few  cocci  were  injected  into  a  vein,  or  when  two 
days  or  more  were  allowed  to  elapse  after  injury  to  the  cardiac 
valves.  Subsequently  Weichselbaum,  Prudden,  and  Frankel  and 
Sanger  obtained  confirmatory  results,  thus  establishing  the  fact  that 
when  the  valves  are  first  injured  mechanically  (or  chemically— 
Prudden)  the  injection  into  a  vein  of  a  pure  culture  of  Staphylococcus 
aureus  gives  rise  to  a  genuine  ulcerative  endocarditis.  It  has  been 
further  shown  by  Ribbert  that  the  same  result  may  be  obtained  with- 
out previous  injury  to  the  valves  by  injecting  into  a  vein  the  staphy- 
lococcus from  a  potato  culture  suspended  in  water.  In  his  experi- 
ments not  only  the  micrococci  from  the  surface  but  the  superficial 
layer  of  the  potato  was  scraped  off  with  a  sterilized  knife  and  mixed 
with  distilled  water  ;  and  the  successful  result  is  ascribed  to  the  fact 
that  the  little  agglomerations  of  micrococci  and  infected  fragments 
of  potato  attach  themselves  to  the  margins  of  the  valves  more  readily 
than  isolated  cocci  would  do.  In  these  experiments  the  mitral  and 
tricuspid  valves  were  affected,  while  the  semilunar  valves  remained 
intact.  In  ulcerative  endocarditis  it  is  evident  that  cocci  detached 
from  the  diseased  valves  must  find  their  way  into  the  circula- 
tion. As  a  matter  of  fact,  masses  of  micrococci  are  carried  away  by 
the  blood  stream  and  form  emboli  in  various  parts  of  the  body,  which 
become  secondary  foci  of  infection  and  give  rise  to  local  necrotic 
changes  and  accumulations  of  pus.  While  this  undoubtedly  occurs, 
it  is  generally  admitted  that  the  mycotic  infection  of  the  cardiac 
valves  is  usually  a  secondary  affection,  resulting  from  the  transpor- 
tation of  micrococci  in  the  blood  current  from  some  other  infected 
focus.  But  there  is  no  general  development  of  micrococci  in  the  cir- 


284  PYOGENIC   BACTERIA. 

culating  fluid,  and  in  man,  as  in  animals  infected  experimentally,  a 
microscopic  examination  of  the  blood  for  microorganisms  usually 
gives  a  negative  result.  Culture  experiments  may,  however,  demon- 
strate their  presence.  Thus  recent  investigations  by  Netter,  Eisel- 
berg,  and  others  show  that  the  pus  cocci  are  usually  present  in  the 
blood  in  small  numbers,  as  demonstrated  by  culture  experiments,  in 
septic  infection  from  wounds. 

2.  STAPHYLOCOCCUS  PYOGENES  ALBUS. 

Isolated  by  Rosenbach  (1884)  from  the  pus  of  acute  abscesses,  in 
which  it  is  sometimes  the  only  microorganism  present,  and  some- 
times associated  with  other  pus  cocci.  In  thirty-three  acute  abscesses 
examined  by  Passet  (1885)  it  was  associated  with  Staphylococcus 
aureus  in  eleven,  with  Staphylococcus  citreus  in  two,  with  Strepto- 
coccus pyogenes  in  one,  with  both  Staphylococcus  citreus  and  Strep- 
tococcus pyogenes  in  one,  and  was  obtained  alone  from  four. 

In  its  morphology  this  micrococcus  is  identical  with  the  preced- 
ing, but  it  is  distinguished  from  it  by  the  absence  of  pigment  and 
by  being  somewhat  less  pathogenic.  Surface  cultures  upon  nutrient 
agar  or  potato  have  a  milk-white  color.  It  liquefies  gelatin  in  the 
same  way  as  does  the  golden  Staphylococcus,  but  the  deposit  at  the 
bottom  of  the  liquefied  gelatin  is  without  color.  In  the  temperature 
conditions  favorable  to  its  growth,  and  in  its  biological  characters 
generally,  with  the  exceptions  noted,  it  is  not  to  be  distinguished 
from  the  species  previously  described.  According  to  Fliigge,  it  is 
more  common  than  aureus  among  many  of  the  lower  animals. 

Pathogenesis. — Fortunati  has  tested  the  comparative  pathogenic 
power  of  Staphylococcus  aureus  and  Staphylococcus  albus  by  inocu- 
lations into  the  cornea  of  rabbits.  A  purulent  infiltration  of  the 
cornea  and  panophthalmitis  resulted  when  Staphylococcus  aureus 
was  inoculated  upon  the  surface  of  the  cornea  by  scratching  with  an 
infected  needle,  but  inoculations  made  in  the  same  way  with  Staphy- 
lococcus albus  healed  spontaneously  or  gave  rise  to  a  perforating 
ulcer.  After  paracentesis  of  the  cornea  with  an  instrument  infected 
with  Staphylococcus  aureus  panophthalmitis  developed  in  thirty  hours; 
the  same  result  occurred  at  the  end  of  sixty  to  seventy-two  hours 
when  the  instrument  was  infected  with  Staphylococcus  albus.  When 
a  sterilized  instrument  was  used  the  result  was  negative.  In  bacteri- 
ological researches  made  by  Gallenga,  in  cases  of  panophthalmitis  in 
man,  Staphylococcus  albus  was  found  in  ten  cultures  and  Staphy- 
lococcus aureus  in  nine. 

Staphylococcus  Epidermidis    Albus   (Welch). 
The  recently  published  researches  of  Welch  show  that  a  white 
Btaphylococcus,   probably   identical    with  Staphylococcus  pyogenes 


PYOGENIC   BACTERIA.  285 

albus  of  Rosenbach,  is  the  most  common  microorganism  upon  the 
surface  of  the  body,  and  that  "  it  is  very  often  present  in  parts  of  the 
epidermis  deeper  than  can  be  reached  by  any  known  means  of  cuta- 
neous disinfection  save  the  application  of  heat."  With  reference  tc 
this  coccus  Welch  says  : 

"So  far  as  our  observations  extend — and  already  they  amount  to  a 
large  number — this  coccus  may  be  regarded  as  a  nearly,  if  not  quite,  con- 
stant inhabitant  of  the  epidermis.  It  is  now  clear  why  I  have  proposed  to 
call  it  the  Staphylococcus  epidermidis  albus.  It  possesses  such  feeble  pyo- 
genic  capacity,  as  is  shown  by  its  behavior  in  wounds  as  well  as  by  experi- 
ments on  rabbits,  that  the  designation  Staphylococcus  pyogenes  albus  does 
not  seem  appropriate.  Still,  I  am  not  inclined  to  insist  too  much  upon  this 
point,  as  very  probably  this  coccus,  which  has  hitherto  been  unquestionably 
identified  by  Bossowski  and  others  with  the  ordinary  Staphylococcus  pyo- 
genes albus  of  Rosenbach,  is  an  attenuated  or  modified  form  of  the  latter 
organism,  although,  as  already  mentioned,  it  presents  some  points  of  differ- 
ence from  the  classical  description  of  the  white  pyogenic  coccus." 

According  to  Welch,  this  coccus  differs  from  Staphylococcus  pyo- 
genes aureus  not  only  in  color,  but  also  in  the  fact  that  it  liquefies 
gelatin  more  slowly,  does  not  so  quickly  cause  coagulation  of  milk, 
and  is  far  less  virulent  when  injected  into  the  circulation  of  rabbits. 
It  has  been  shown  by  the  researches  of  Bossowski  and  of  Welch 
that  this  coccus  is  very  frequently  present  in  aseptic  wounds,  and 
that  usually  it  does  not  materially  interfere  with  the  healing  of 
wounds,  although  sometimes  it  appears  to  cause  suppuration  along 
the  drainage  tube,  and  it  is  the  usual  cause  of  "  stitch  abscess." 
Bossowski,  in  fifty  cases  of  wounds  treated  antiseptically,  obtained 
bacteria  from  the  discharges  in  forty,  and  in  twenty-six  of  these 
cases  he  found  Staphylococcus  pyogenes  albus  ;  Staphylococcus  au- 
reus was  found  nine  times,  Streptococcus  pyogenes  in  two,  and  vari- 
ous non-pathogenic  bacteria  in  eight.  In  forty-five  laparotomy 
wounds  examined  by  Ghrisky  and  Robb,  in  which  strict  antiseptic 
precautions  had  been  observed,  bacteria  were  found  in  thirty-one,  and 
in  nineteen  of  this  number  Staphylococcus  albus  was  present, 
Staphylococcus  aureus  in  five,  Bacillus  coli  communis  in  six,  and 
Streptococcus  pyogenes  in  three. 

3.      STAPHYLOCOCCUS  PYOGENES   CITREUS. 

Isolated  by  Passet  (1885)  from  the  pus  of  acute  abscesses.  In  thirty - 
three  cases  examined  it  was  found  associated  with  Staphylococcus  albus  in 
two  and  with  Staphylococcus  albus  and  Streptococcus  pyogenes  in  one. 

In  its  morphology  this  coccus  is  identical  with  the  two  preceding  species, 
from  which  it  is  distinguished  by  the  formation  of  a  lemon-yellow  pigment, 
instead  of  a  golden  or  orange-yellow  as  in  Staphylococcus  aureus.  The 
pigment  is  only  formed  in  the  presence  of  oxygen.  This  coccus  is  said  by 
Frankel  to  liquefy  gelatin  more  slowly  than  the  previously  described  species 
— Staphylococcus  aureus  and  Staphylococcus  albus. 

As  to  its  pathogenic  properties  we  have  no  definite  information.  It  is 
included  among  the  pyogenic  bacteria  because  of  its  occasional  presence  in 


286  PYOGENIC   BACTERIA. 

the  pus  of  acute  abscesses,  although  it  has  heretofore  only  been  found  in  as- 
sociation with  other  microorganisms. 

4.     MICROCOCCUS  PYOGENES  TENUIS. 

Obtained  by  Rosenbach  (1884)  from  pus  in  three  cases  out  of  thirty- nine 
examined. 

Morphology.—  Micrococci,  somewhat  irregular  in  size,  but  larger  than 
Staphylococcus  albus,  and  seldom  associated  in  masses.  Frequently  the  in- 
dividual cocci  present  the  appearance  of  consisting  of  two  deeply  stained 
masses  separated  from  each  other  by  a  paler  interspace.  Cultures  upon  the 
surface  of  nutrient  agar  form  a  very  thin,  transparent  layer  of  about  one 
millimetre  in  breadth  along  the  line  of  inoculation ;  this  resembles  a  thin 
layer  of  varnish. 

Pathogenesis  undetermined.  (Micrococcus  pneumonias  crouposae  ?) 

5.    STREPTOCOCCUS  PYOGENES. 

Synonyms. — Micrococcus  of  erysipelas  (Fehleisen) ;  Streptococcus 
erysipelatos  ;  Streptococcus  of  pus  ;  Streptococcus  longus  (Von  Lin- 
gelsheim). 

Obtained  by  Fehleisen  from  the  skin  involved  in  cases  of  erysipe- 
las (1883),  and  by  Rosenbach  (1884)  and  Passet  (1885)  from  the  pus 
of  acute  abscesses.  The  characters  of  the  "  streptococcus  of  erysipe- 
las "  of  Fehleisen  and  the  "  Streptococcus  pyogenes  "  of  Rosenbach 
and  Passet  are  generally  admitted  to  be  identical,  although  some 
bacteriologists  still  describe  them  separately  and  cultures  from  the 
two  sources  are  still  retained  in  bacteriological  laboratories  under  the 
names  originally  given  them. 

Rosenbach  found  Streptococcus  pyogenes  alone  in  fifteen  cases, 
and  associated  with  staphylococci  in  five  cases,  out  of  thirty-nine 
cases  examined  of  acute  pus  formation.  Passet,  in  thirty-three 
similar  cases,  obtained  the  streptococcus  alone  in  eight  and  associated 
with  staphylococci  in  two.  Subsequent  researches  show  that  this 
micrococcus  is  frequently,  if  not  constantly,  present  in  puerperal 
metritis  ;  that  it  is  the  most  frequent  microorganism  associated  with 
ulcerative  endocarditis  ;  that  it  is  frequently  present  in  diphtheritic 
false  membranes,  and  especially  in  those  cases  of  diphtheritic  inflam- 
mation which  are  secondary  to  scarlet  fever  and  measles  (Prudden). 
Numerous  investigations  made  by  bacteriologists  during  the  past  few 
years  indicate  that  this  is  a  very  important  and  widely  distributed 
pathogenic  microorganism.  It  has  also  been  frequently  found  upon 
exposed  mucous  surfaces — mouth,  nose,  vagina — of  healthy  in- 
dividuals. 

According  to  the  researches  (1891)  of  Von  Lingelsheim,  the  Strep- 
tococcus pyogenes  differs  from  Streptococcus  erysipelatos  in  be- 
ing pathogenic  both  for  mice  and  rabbits,  while  the  latter  is  patho- 


PYOGENIC   BACTERIA.  287 

genie  for  rabbits  only.  The  author  named,  as  a  result  of  extended  and 
carefully  conducted  comparative  studies,  arrives  at  the  following 
conclusions : 

"  According- to  my  observations,  there  are  two  great  groups  among  the 
streptococci.  These  cannot  be  distinguished  one  from  the  other  in  cultures 
in  highly  albuminous  media  (pus,  blood  serum),  but  present  constant  dif- 
ferences when  cultivated  in  bouillon.  The  decisive  characteristics  in  this 
medium  are  :  macroscopic,  the  cloudiness  of  the  medium  ;  microscopic,  the 
length  of  the  chains.  The  two  groups  are  with  difficulty  distinguished  in 
agar  cultures ;  more  easily  in  gelatin,  in  which  the  streptococcus  which 
forms  short  chains  causes  a  slight  liquefaction,  while  the  Streptococcus 
longus  does  not.  Upon  potato  Streptococcus  brevis  alone  shows  a  visible 
growth.  .  .  .  We  see  here  a  group  of  streptococci  which  we  separate  from 
the  others,  because  of  their  microscopic  and  cultural  differences,  under  the 
name  of  Streptococcus  brevis,  which  is  also  distinguished  by  having  no 
pathogenic  action  upon  the  animals  usually  experimented  upon.  We 
recognize,  on  the  other  hand,  the  streptococci  which  we  have  grouped  to- 
gether as  Streptococcus  longus  as  all  pathogenic  and  about  in  equal  degree 
for  a  certain  species  of  animal  (rabbits) ;  but  by  experiments  upon  other 
species  (mice)  we  arrive  at  the  conclusion  that  there  must  also  be  differences 
between  these  streptococci.  It  appears  that  the  streptococci  which  are  dis- 
tinguished by  their  high  degree  of  pathogenic  power  upon  mice  are  also 
those  which  are  distinguished  in  bouillon  cultures  by  the  formation  of  con- 
glomerate masses.  We  find  among  these  also  one  which  is  distinguished 
by  especial  virulence  for  mice,  and  that  this  one  is  distinguished  in  cultures 
by  its  scanty  growth  upon  ox  serum." 

The  more  recent  researches  of  Knorr  (1893),  and  of  Waldvogel 
(1894),  indicate  that  the  classification  of  the  streptococci  proposed  by 
von  Lingelsheim  has  no  great  value,  and  show  that  marked  changes 
in  biological  characters  and  in  pathogenic  power  may  result  from 
cultivation  in  special  media,  or  from  successive  inoculations  into 
animals. 

Morphology. — Spherical  cocci,  from  0.4  yu  to  1  /f  in  diameter,  but 
varying  considerably  in  dimensions  in  different  cultures,  and  even 
in  a  single  chain.  Multiply  by  binary  division, 
in  one  direction  only,  forming  chains,  in  which 
the  elements  are  commonly  associated  in  pairs. 
Under  certain  circumstances,  instead  of  form- 
ing chains,  a  culture  may  contain  only,  or 
chiefly,  diplococci ;  but  usually  chains  contain- 
ing from  four  to  twenty  or  more  elements  are 
formed,  and  these  are  frequently  associated 
in  tangled  masses.  Occasionally  one  or  more 
cells  in  a  chain  greatly  exceed  their  fellows  in 
size,  and  some  bacteriologists  suppose  that  Fl°-  82.— PUS  containing 

n        , .  ,1  streptococci.        X  800. 

these  cells  serve  as  reproductive  spores — arthro-          (Fiugge.) 
spores — but  this  has  not  been  definitely  proven. 

Stains  readily  with  the  aniline  colors  and  by  Gram's  method. 


288 


PYOGENIC  BACTERIA. 


Biological  Characters. — Grows  readily  in  various  liquid  and 
solid  culture  media,  including  all  of  those  usually  employed  in  bac- 
teriological researches.  The  most  favorable  temperature  for  its  de- 
velopment is  from  30°  to  37°  C.,  but  it  multiplies  freely  at  the  ordi- 
nary room  temperature — 16°  to  18°  C. 

Streptococcus  pyogenes  is  a  facultative  anaerobic,  growing 
both  in  the  presence  and  absence  of  oxygen.  It 
does  not  liquefy  gelatin,  and  in  gelatin  stick 
cultures  it  grows  along  the  line  of  puncture, 
forming  numerous  small,  spherical,  translu- 
cent, whitish  colonies,  which  are  closely  crowd- 
ed together  at  the  upper  portion  of  the  line  of 
growth,  and  often  distinctly  separated  from 
each  other  below ;  upon  the  surface  there  is 
often  no  growth,  or  a  scanty  development  may 
occur  about  the  point  of  entrance  of  the  inocu- 
lating needle.  The  minute  colonies  along  the 
line  of  puncture  are  already  visible  at  the  end 
of  twenty-four  hours  in  cultures  kept  in  the 
incubating  oven  at  30°  to  35°  C.,  and  at  the  end 
of  three  or  four  days  they  have  reached  their 
full  development,  forming  a  semi -opaque,  white, 
granular  column,  upon  the  margins  of  which 
the  separate  colonies  are  seen  projecting  into  the 
gelatin.  On  gelatin  plates  very  small,  translu- 
cent colonies  are  developed,  which  upon  the  sur- 
face spread  out  to  form  a  flat,  transparent  disc 
of  about  one-half  millimetre.  Under  a  low  mag- 
nifying power  these  colonies  are  seen  to  be  slight- 
ly granular  and  have  a  yellowish  color.  At  a 
later  date  they  become  darker  and  less  trans- 
parent, and  the  margin  may  show  irregular  projections  made  up  of 
tangled  masses  of  cocci  in  chains.  The  characters  of  growth  in 
nutrient  agar  and  in  jellified  blood  serum  are  similar  to  those  in  gela- 
tin, and  on  agar  plates  colonies  are  formed  similar  to  those  above 
described,  except  that  they  are  somewhat  smaller  and  more  trans- 
parent. Fehleisen  and  De  Simone  state  that  the  erysipelas  coccus 
may  develop  upon  the  surface  of  cooked  potato,  but  most  authorities 
— Fliigge,  C.  Frankel,  Passet,  Baumgarten — agree  that  no  growth 
occurs  upon  potato.  Milk  is  a  favorable  medium  for  the  growth  of 
this  micrococcus,  and  the  casein  is  coagulated  by  it.  A  slightly  acid 
reaction  of  the  culture  medium  does  not  prevent  its  development. 
The  thermal  death-point,  as  determined  by  the  writer,  is  between 
52°  and  54°  C. ,  the  time  of  exposure  being  ten  minutes.  According 


Fio.  83.— Streptococcus 
of  erysipelas  in  nutrient 
gelatin;  stick  culture  at 
end  of  four  days  at  16°- 
18°  C.  (Baumgarten). 


PYOGENIC   BACTERIA.  289 

to  De  Simone,  a  temperature  of  39.5°  to  41°  C.  maintained  for  two 
days  is  fatal  to  this  micrococcus. 

Manfred!  and  Traversa  have  injected  filtered  cultures  into  frogs, 
guinea-pigs,  and  rabbits  for  the  purpose  of  ascertaining  if  any  solu- 
ble toxic  substance  is  produced  during  the  growth  of  Streptococcus 
pyogenes.  They  report  that  in  some  cases  convulsions  and  in  others 
paralysis  resulted  from  these  injections. 

Von  Lingelsheim  has  (1891)  reported  the  following  results 
obtained  in  an  extended  series  of  experiments  made  to  determine 
the  germicidal  power  of  various  chemical  agents  as  tested  upon 
this  microorganism — tune  of  exposure  two  hours  :  Hydrochloric  acid 
1  : 250,  sulphuric  acid  1  : 250,  caustic  soda  1  : 130,  ammonia  1  : 25, 
mercuric  chloride  1  : 2,500,  sulphate  of  copper  1  : 200,  chloride  of 
iron  1  :  500,  terchloride  of  iodine  1  :  750,  peroxide  of  hydrogen  1  :  50, 
carbolic  acid  1  : 300,  cresol  1  : 250,  lysol  1  :  300,  creolin  1  : 130,  naph- 
thylamin  1  : 125,  malachite  green  1  :  3,000,  pyoktanin  1  :  700. 


FIG.  84.— Section  from  margin  of  an  erysipelatous  inflammation,  showing  streptococci  in 
lymph  spaces.  From  a  photograph  by  Koch.  X  900. 

Pathogenesis. — When  inoculated  into  the  cornea  of  rabbits 
Streptococcus  pyogenes  gives  rise  to  keratitis.  Inoculations  into  the 
ear  of  the  same  animal  usually  give  rise  to  a  localized  erysipelatous 
inflammation  accompanied  by  an  elevation  of  temperature  in  the  in- 
oculated ear ;  at  the  end  of  thirty-six  to  forty-eight  hours  the  in- 
flamed area,  which  has  well-defined  margins  and  a  bright-red  color, 
extends  from  the  point  of  inoculation  along  the  course  of  the  veins  to 
the  root  of  the  ear.  This  appearance  passes  away  in  the  course  of  a 
few  days  and  the  animal  recovers.  Subcutaneous  injections  into  mice 
or  rabbits  are  usually  without  result,  and  the  last-named  animal  also 
withstands  injections  of  considerable  quantities  into  the  general  cir- 
culation through  a  vein.  When,  however,  the  animal  has  previously 
been  weakened  by  the  injection  of  toxic  substances  the  streptococcus 
may  multiply  in  its  body  and  cause  its  death  (Fliigge). 

Fehleisen  has  inoculated  cultures,  obtained  in  the  first  instance 
from  the  skin  of  patients  with  erysipelas,  into  patients  in  hospital 
suffering  from  lupus  and  carcinoma,  and  has  obtained  positive  re- 
sults, a  typical  erysipelatous  inflammation  having  developed 
19 


PYOGENIC   BACTERIA. 

around  the  point  of  inoculation  after  a  period  of  incubation  of  from 
fifteen  to  sixty  hours.  This  was  attended  with  chilly  sensations  and 
an  elevation  of  temperature.  Persons  who  had  recently  recovered 
from  an  attack  of  erysipelas  proved  to  be  immune. 

Sections  made  from  the  ear  of  an  inoculated  rabbit,  or  of  skin  taken 
from  the  affected  area  in  erysipelas  in  man,  show  the  streptococci  in 
considerable  numbers  in  the  lymph  channels,  but  not  in  the  blood 
vessels.  They  are  more  numerous,  according  to  Koch  and  to  Fehl- 
eisen,  upon  the  margins  of  the  erysipelatous  area,  and  may  even  be 
seen  in  the  lymph  channels  a  little  beyond  the  red  margin  which 
marks  the  line  of  progress  of  the  infection. 

The  researches  of  Weichselbaum  and  others  show  that  Strepto- 
coccus pyogenes  is  the  infecting  microorganism  in  a  certain  propor- 
tion of  the  cases  of  ulcerative  endocarditis.  The  author  named 
found  it  in  four  cases  out  of  fifteen  examined,  and  in  two  cases  of 
endocarditis  verrucosa  out  of  thirteen.  In  a  previously  reported  series 
of  sixteen  cases  (fourteen  of  ulcerative  endocarditis  and  two  of  ver- 
rucosa) the  streptococcus  was  found  in  six. 

In  diphtheritic  false  membranes  this  streptococcus  is  very  com- 
monly present,  and  in  certain  cases  attended  with  a  diphtheritic  exu- 
dation, in  which  the  Bacillus  diphtherias  has  not  been  found  by  com- 
petent bacteriologists,  it  seems  probable  that  Streptococcus  pyogenes 
is  the  pathogenic  microorganism  responsible  for  the  local  inflamma- 
tion and  its  results.  Thus  in  a  series  of  twenty-four  cases  studied  by 
Prudden  in  1889  the  bacillus  of  Loifler  was  not  found,  "but  a  strep- 
tococcus apparently  identical  with  Streptococcus  pyogenes  was  found 
in  twenty-two."  Chantemesse  and  Widal  have  also  reported  cases 
in  which  a  fibrinous  exudate  resembling  that  of  diphtheria  was  as- 
sociated with  a  streptococcus.  "  These  forms  of  so-called  diphtheria 
are  most  commonly  associated  with  scarlatina  and  measles,  erysipe- 
las, and  phlegmonous  inflammation,  or  occur  in  individuals  exposed 
to  these  diseases  ;  but  whether  exclusively  under  these  conditions  is 
not  yet  established  "  (Prudden). 

Loffler  has  described  under  the  name  of  Streptococcus  articu- 
lorum  a  micrococcus  obtained  by  him  from  the  affected  mucous 
membrane  in  cases  of  diphtheria,  and  which  he  believes  to  be  acci- 
dentally present  and  without  any  etiological  import  in  this  disease. 
In  its  characters  it  closely  resembles  Streptococcus  pyogenes  and  is 
perhaps  a  variety  of  this  widely  distributed  species.  Its  characters 
are  described  by  Flugge  as  follows  : 

44  Cultivated  in  nutrient  gelatin,  it  forms  at  the  end  of  three  days  small, 
transparent,  lijrht-tfray  drops,  upon  the  margin  of  which,  under  tlio  micro- 
scope, the  cocci  in  twisted  chains  may  be  observed.  As  many  as  one  hun- 


PYOGENIC   BACTERIA.  291 

dred  elements  may  be  found  in  a  single  chain,  and  some  of  these  are  distin- 
guished by  their  size ;  occasionally  whole  chains  are  made  up  of  these  large 
cocci,  and  when  closely  observed  some  of  these  may  present  indications  of 
division  transversely  to  the  axis  of  the  chain.  Subcutaneous  inoculation  of 
cultures  into  mice  results  in  the  death  of  a  considerable  number  of  these  ani- 
mals—more than  half ;  and  the  streptococci  are  found  in  the  spleen  and  other 
organs.  Inoculation  into  the  ear  of  rabbits  causes  an  erysipelatous  inflam- 
mation. When  injected  into  the  circulation  of  these  animals  through  a  vein 
joint  affections  are  developed  in  from  four  to  six  days,  and  a  purulent  ac- 
cumulation occurs  in  which  the  streptococci  are  found.  In  two  rabbits  in- 
oculated in  the  same  way  with  a  culture  of  the  streptococcus  of  erysipelas, 
Loffler  has  observed  a  similar  result." 

Recent  researches  indicate  that  infection  by  Streptococcus  pyo- 
genes  through  the  endometrium  is  the  usual  cause  of  puerperal 
fever.  Thus  Clivio  and  Monti  demonstrated  its  presence  in  five 
cases  of  puerperal  peritonitis.  Czerniewski  found  it  in  the  lochia  of 
a  large  number  (thirty-five  out  of  eighty-one)  of  women  suffering 
from  puerperal  fever,  but  in  the  lochia  of  fifty-seven  healthy  puer- 
peral women  he  was  only  able  to  find  it  once.  In  ten  fatal  cases  he 
found  it  in  every  instance,  both  in  the  lochial  discharge  during  life 
and  in  the  organs  after  death.  Widal  carefully  studied  a  series  of 
sixteen  cases  and  arrived  at  the  conclusion  that  this  was  the  infect- 
ing microorganism  in  all.  Bumm  and  other  observers  have  given 
similar  evidence.  Eiselsberg  and  Emmerich  have  succeeded  in  de- 
monstrating the  presence  of  the  streptococcus  in  hospital  wards  con- 
taining cases  of  erysipelas.  That  puerperal  fever  may  result  from 
infection  through  the  finger  of  the  accoucheur,  when  he  has  previ- 
ously been  in  contact  with  cases  of  erysipelas,  has  long  been  taught, 
and,  in  view  of  the  facts  above  recorded,  is  not  difficult  to  under- 
stand. But  in  view  of  the  fact  that  the  streptococcus  of  pus  .has  been 
found  in  vaginal  mucus  and  in  the  buccal  and  nasal  secretions  of 
healthy  persons,  it  may  appear  strange  that  cases  of  puerperal  fever 
not  traceable  to  infection  from  erysipelas  or  from  preceding  cases 
do  not  occur  more  frequently.  This  is  probably  largely  due  to  an 
attenuation  of  the  pathogenic  power  of  the  streptococcus  when  it 
leads  a  saprophytic  existence.  Widal  asserts  that,  when  cultivated 
in  artificial  media  for  a  few  weeks,  the  cultures  no  longer  have  their 
original  virulence,  and  Bumm  has  made  the  same  observation.  On 
the  other  hand,  in  "streptococcus-peritonitis"  occurring  as  a  result 
of  puerperal  infection  Bumm  states  that  the  thin,  bright-yellow, 
odorless  fluid  contained  in  the  cavity  of  the  abdomen  is  extremely 
virulent ;  a  very  slight  trace,  a  fragment  of  a  drop,  injected  into  the 
abdominal  cavity  of  a  rabbit,  is  sufficient  within  twenty-four  hours 
to  cause  a  general  septic  inflammation  with  a  bloody  serous  exuda- 
tion, quickly  terminating  in  the  death  of  the  animal ;  injected  sub- 
cutaneously  it  gives  rise  to  an  enormous  phlegmon  which  also 


292  PYOGENIC  BACTERIA. 

quickly  proves  fatal.  But  cultures  of  Streptococcus  pyogenes,  after 
it  has  been  carried  through  successive  generations  in  artificial  media, 
injected  beneath  the  skin  of  a  rabbit,  usually  produce  no  result,  or 
at  most  an  abscess  of  moderate  dimensions. 

It  seems  probable  that  the  micrococcus  isolated  by  Fliigge  from 
necrotic  foci  in  the  spleen  of  a  case  of  leucocythsemia,  and  described 
by  him  under  the  name  of  Streptococcus  pyogenes  malignus,  was 
simply  a  very  pathogenic  variety  of  the  streptococcus  of  pus.  He 
was  not  able  to  differentiate  it  from  Streptococcus  pyogenes  by  its 
morphology  or  growth  in  culture  media,  but  it  proved  far  more 
pathogenic  when  tested  upon  animals.  Mice  inoculated  subcutane- 
ously  with  a  minute  quantity  of  a  pure  culture  died,  without  excep- 
tion, in  three  to  five  days.  A  large  abscess  was  formed  at  the  point 
of  inoculation,  and  the  blood  of  the  animal  contained  numerous  cocci 
in  pairs  and  chains.  Rabbits  inoculated  in  the  ear  showed  at  first 
the  same  local  appearances  as  result  from  inoculations  with  strepto- 
coccus of  pus  and  of  erysipelas,  but  after  two  or  three  days  symp 
toms  of  general  infection  were  developed,  and  death  occurred  at  the 
end  of  three  or  four  days.  At  the  autopsy  the  cocci  were  found  in 
the  blood,  and  frequently  there  were  purulent  collections  in  the 
joints  containing  the  same  microorganism.  Krause  has  also  de- 
scribed a  streptococcus  which  only  differs  from  Streptococcus  pyo- 
genes of  Rosenbach  and  Passet  by  the  greater  virulence  manifested 
by  its  cultures. 

The  fact  that  pathogenic  bacteria  may  attain  an  intensified  de- 
gree of  virulence  by  cultivation  in  the  bodies  of  susceptible  animals 
was  demonstrated  by  Davaine  many  years  ago,  and  is  fully  estab- 
lished by  the  experiments  of  Pasteur  and  others.  It  is  true  of  the 
anthrax  bacillus,  of  the  writer's  Micrococcus  Pasteuri,  and  of  other 
well-known  pathogenic  microorganisms.  The  reverse  of  this — at- 
tenuation of  virulence  as  a  result  of  cultivation  in  artificial  media— 
is  also  well  established  for  several  pathogenic  species.  Now  it 
appears  that  the  attenuated  streptococcus  is  far  less  likely  to  give 
rise  to  erysipelas  or  to  puerperal  infection  than  is  the  same  micro- 
organism as  obtained  from  a  case  of  one  or  the  other  of  these  infec- 
tious diseases.  The  same  is  probably  true  also  of  Staphylococcus 
aureus  and  other  facultative  parasites  which  are  found  as  sapro- 
phytes upon  the  surface  of  the  body  and  upon  exposed  mucous  mem- 
branes in  healthy  persons.  And  it  is  not  improbable  that  attenuated 
varieties  of  these  micrococci  which  find  their  way  into  open  wounds, 
or  into  the  uterine  cavity  shortly  after  parturition,  if  they  escape 
destruction  by  the  sanguineous  discharge,  acquire  increased  patho- 
genic power  from  their  multiplication  in  it,  as  a  result  of  which  they 
aiv  al)lo  to  invade  tin-  living  tissues.  But  it  appears  probable  that 


PYOGENIC   BACTERIA.  293 

infection  through  open  wounds  does  not  depend  alone  upon  the 
potency  of  the  pathogenic  micrococci  present  in  them,  but  also  upon 
the  absorption  of  chemical  poisons  produced  by  septic  (putrefactive) 
bacteria,  which  weaken  the  vital  resisting  power  of  the  tissues. 
Gottstein,  as  a  result  of  experiments  made  by  him,  is  of  the  opinion 
that  the  resorption  of  broken-down  red  blood  corpuscles  favors  infec- 
tion by  pathogenic  bacteria  present  in  wounds  ;  and  he  has  shown 
that  the  injection  into  animals  of  certain  toxic  substances  which  de- 
stroy the  red  corpuscles  in  the  circulation  makes  them  susceptible  to 
the  pathogenic  action  of  certain  bacteria  which  are  harmless  for 
them  under  ordinary  circumstances.  Thus  a  guinea-pig,  an  animal 
which  is  immune  against  the  bacillus  of  fowl  cholera,  succumbed  to  an 
inoculation  made  after  first  injecting  subcutaneously  0.06  gramme  of 
hydracetin  dissolved  in  alcohol.  At  the  autopsy  hsemorrhagic  exu- 
dations were  found  in  the  serous  cavities,  haemorrhagic  infarctions 
in  the  lungs,  and  quantities  of  the  bacillus  injected  were  found  in 
the  blood  and  in  fluid  from  the  cavity  of  the  abdomen. 

In  man  the  ever-present  pus  cocci  are  more  likely  to  invade  the 
tissues,  forming  furuncles,  carbuncles,  and  pustular  skin  eruptions, 
or  erysipelatous  and  phlegmonous  inflammations,  when  the  standard 
of  health  is  reduced  from  any  cause,  and  especially  when  by  absorp- 
tion or  retention  various  toxic  organic  products  are  present  in  the 
body  in  excess.  It  is  thus  that  we  would  explain  the  liability  to  these 
local  infections,  as  complications  or  sequelse  of  various  specific  infec- 
tious diseases,  in  the  victims  of  chronic  alcoholism,  in  those  exposed 
to  septic  emanations  from  sewers,  etc. ,  and  probably  in  many  cases 
from  the  absorption  of  toxic  products  formed  in  the  alimentary  canal 
as  a  result  of  the  ingestion  of  improper  food,  or  of  abnormal  fermen- 
tative changes  in  the  contents  of  the  intestine,  or  from  constipation. 

The  Pus  Cocci  in  Inflammations  of  Mucous  Membranes. — 
To  what  extent  the  pus  cocci  are  responsible  for  inducing  and  main- 
taining non-specific  inflammations  of  mucous  membranes  has  not 
been  determined  ;  but  having  demonstrated  the  pyogenic  properties 
of  these  cocci,  their  presence  in  the  purulent  discharges  from  inflamed 
mucous  membranes  can  scarcely  be  considered  as  unimportant,  not- 
withstanding the  fact  that  they  are  also  frequently  found  in  secre- 
tions from  healthy  mucous  surfaces.  They  are  likewise  found  upon 
the  skin  of  healthy  persons,  and  yet  we  have  unimpeachable  experi- 
mental evidence  that  they  may  produce  a  local  inflammation,  at- 
tended with  pus  formation,  when  injected  subcutaneously,  or  even 
when  freely  applied  to  the  uninjured  surface. 

In  otitis  media  Levy  and  Schrader  obtained  Staphylococcus 
albus  in  pure  cultures  in  three  cases  out  of  ten  in  which  paracentesis 
was  performed,  and  in  two  others  it  was  present  in  association  with 


294  PYOGENIC   BACTERIA. 

other  microorganisms.  In  eighteen  cases  of  otitis  media  in  young 
children  Netter  found  Staphylococcus  aureus  six  times  and  Strepto- 
coccus pyogenes  thirteen  times.  Scheibe,  in  eleven  cases  in  which 
perforation  had  not  yet  taken  place,  found  Staphylococcus  albus  in 
two  and  various  other  microorganisms  in  the  remaining  cases  ;  Sta- 
phylococcus aureus  was  not  present  in  any.  Habermann  obtained 
aureus  associated  with  other  bacteria  in  a  single  case  of  purulent 
otitis  media.  In  a  series  of  eight  cases  occurring  as  a  sequela  of 
influenza  Scheibe  obtained  Streptococcus  pyogenes  in  two,  "  diplo- 
coccus  pneumonias "  in  two,  Staphylococcus  aureus  in  one,  Strepto- 
coccus pyogenes  and  Staphylococcus  albus  together  in  two,  and  Strep- 
tococcus pyogenes  in  association  with  an  undescribed  micrococcus  in 
one.  In  all  of  these  cases  a  slender  bacillus  was  also  present,  as 
shown  by  microscopical  examination,  which  did  not  grow  in  any  of 
the  culture  media  employed.  Bordoni-Uffreduzzi  and  Gradenigo 
have  tabulated  the  results  obtained  by  various  bacteriologists  who 
have  examined  pus  obtained  through  the  previously  intact  tympanic 
membrane.  In  thirty-two  cases  of  this  character  the  microorganism 
most  frequently  found  was  diplococcus  pneumonias  (Micrococcus 
pneumonias  crouposas  of  the  present  writer),  which  was  present  in  a 
pure  culture  in  thirteen  and  associated  with  Staphylococcus  aureus 
in  one,  with  Staphylococcus  albus  in  one,  and  with  Streptococcus 
pyogenes  in  one.  In  the  other  sixteen  cases  the  pyogenic  cocci  were 
present  in  all  but  two,  in  which  bacilli  were  found — Bacillus  tenuis 
in  one,  a  non-liquefying  bacillus  in  one.  In  twenty-seven  cases  in 
which  the  pus  was  withdrawn  from  one  to  thirty  days  after  paracen- 
tesis  or  spontaneous  rupture  of  the  membrane,  the  pyogenic  cocci 
were  present  in  twenty  and  diplococcus  pneumonias  in  seven. 

In  acute  nasal  catarrh  Paulsen  found  Staphylococcus  aureus  in 
seven  cases  out  of  twenty-four  examined,  and  E.  Frankel  in  two  out  of 
four  ;  but  it  must  be  remembered  that  Von  Besser  has  shown  that  this 
micrococcus  is  frequently  present  in  the  secretions  from  the  healthy 
nasal  mucous  membrane,  and  we  have  experimental  evidence  that 
the  pus  organisms,  when  introduced  into  the  conjunctival  sac  of 
rabbits  (Widmark),  do  not  give  rise  to  catarrhal  inflammation.  On 
the  other  hand,  Widmark  found  that  when  inoculated  into  the  cornea 
of  rabbits  an  intense  conjunctivitis  resulted,  together  with  keratitis 
and  perforation  of  the  cornea  in  fifteen  per  cent  of  the  cases.  The 
same  author  in  his  bacteriological  researches  obtained  the  pyogenic 
staphylococci  from  the  circumscribed  abscesses  of  blepharadenitis, 
while  in  inflammation  of  the  lacrymal  sac  Streptococcus  pyogenes 
was  usually  present. 

Shougolowicz,in  the  bacteriological  examination  of  twenty-six  cases 
of  trachoma,  found  Staphylococcus  albus  in  twelve,  Staphylococcus 


PYOGENIC   BACTERIA.  295 

aureus  in  nine,  Staphylococcus  citreus  in  three,  and  Staphylococcus 
cereus  albus  in  three.  These  pus  organisms  were  in  a  number  of 
the  cases  associated  with  other  well-known  saprophytes,  and  in  seven 
cases  a  short  bacillus  not  previously  described  was  found.  That 
various  bacilli  are  found  in  the  conjunctival -sac  of  healthy  eyes 
and  in  different  forms  of  conjunctivitis  has  been  shown  by  Fick, 
whose  results  do  not  correspond  in  this  respect  with  those  of  Gif- 
ford,  who  found  almost  exclusively  micrococci.  Whatever  may  be 
the  final  conclusion  as  to  the  role  of  the  pus  cocci  heretofore  de- 
scribed in  the  etiology  of  acute  or  chronic  conjunctivitis,  there  can  be 
no  doubt  of  the  power  of  the  "  gonococcus"  to  induce  a  virulent  in- 
flammation of  the  conjunctivas  when  introduced  into  healthy  eyes. 


6.     MICROCOCCUS   GONORRHCE^E 

Synonym.  —  Gonococcus  (Neisser). 

Discovered  by  Neisser  (1879)  in  gonorrhoeal  pus  and  described  by 
him  under  the  name  of  "  Gonococcus."    Cultivated  by  Bumm  (1885), 
and  infective  virulence  proved  by  inocula- 
tion into  man.    Constantly  present  in  viru- 
lent gonorrhoeal  discharges,  for  the  most 
part  in  the  interior  of  the  pus  cells  or  at- 
tached to  the  surface  of  epithelial  cells. 

Morphology.  —  Micrococci,  usually  join- 
ed in  pairs  or  in  groups  of  four,  in  which 
the  elements  are  flattened  —  "  biscuit- 
shaped."  The  flattened  surf  aces  face  each 
other  and  are  separated,  in  stained  pre- 
parations,  by  an  Unstained  interspace.  c 

KT     ,.  •   A  j     •    •       /11          Fia   S5-—  °»   gonococci  from    a 

Ine  diameter  or  an  associated  pair  or  cells  pure  culture,  x  about  1,000  ;  z>,  gono- 
varies  from  0.8  to-  1.6  w  in  the  long  dia-  cocci  in  PUS  cells  and  epithelial  c*n 

,      _  from  case  of  gonorrhoeal   ophthal- 

meter  —  average  about  1.25  jw—  and   from  mia;  c,  form  and  mode  of  division 


0.6  to  0.8  >u  in  the  line  of  the  interspace  of  gonococci-schematic. 
between  the  biscuit-shaped  elements,  which 

sometimes  present  a  slight  concavity  of  the  flattened  surfaces.  Mul- 
tiplication occurs  alternately  in  two  planes,  and  as  a  result  of  this 
groups  of  four  are  frequently  observed.  But  diplococci  are  more 
numerous  and  are  considered  as  the  characteristic  mode  of  grouping. 
Single,  spherical,  undivided  cells  are  rarely  seen. 

It  must  be  remembered  that  the  morphology  of  this  micrococcus 
as  above  described  does  not  suffice  to  distinguish  it,  for  Bumm  has 
shown  that  "  the  biscuit  form  is  not  at  all  specific  for  the  gonococcus, 
but  is  shared  with  it  by  a  number  of  microorganisms,  which  consist 
of  two  hemispherical  elements  with  the  flattened  surfaces  facing  each 


296  PYOGENIC   BACTERIA. 

other  and  separated  by  a  cleft,  and  some  of  these  correspond  in  their 
morphology,  in  every  detail,  with  the  gonococcus." 

Stains  quickly  with  the  basic  aniline  colors,  especially  with 
methyl  violet,  gentian  violet,  and  f uchsin ;  not  so  quickly  with 
methylene  blue,  which  is,  however,  one  of  the  most  satisfactory 
staining  agents  for  demonstrating  its  presence  in  pus.  Beautiful 
double-stained  preparations  may  be  made  irom  gonorrhoeal  pus, 
spread  upon  a  cover  glass  and  "  fixed/'  secundum  artem,  by  the  use 
of  methylene  blue  and  eosin.  Does  not  stain  by  Gram's  method— 
i.e.,  the  cocci  are  decolorized,  after  having  been  stained  with  an  ani- 
line color,  by  being  immersed  in  the  iodine  solution  employed  in 
Gram's  method  of  staining.  But  this  character  cannot  be  depended 
upon  alone  for  establishing  the  diagnosis,  for  Bumm  has  shown  that 


Fia.  86.—"  Gonococcus  "  in  gonorrhoeal  pus.   From  a  photomicrograph  by  Frankel  and  Pfeiffer. 
X  1,000. 

other  diplococci  are  occasionally  found  in  gonorrhoeal  pus  which  do 
not  stain  by  this  method.  It  serves  to  distinguish  them,  however,  from 
the  common  pus  cocci  heretofore  described — Staphylococcus  aureus, 
Staphylococcus  albus,  Staphylococcus  citreus — which  retain  their 
color  when  treated  in  the  same  way.  A  more  trustworthy  diagnostic 
character  is  that  these  biscuit-shaped  diplococci  are  found  within  the 
pus  cells,  sometimes  one  or  two  pairs  only,  but  more  frequently  in 
considerable  numbers,  and  occasionally  iii  such  numbers  as  to  com- 
pletely fill  the  cell.  No  similar  picture  is  presented  by  pus  from  any 
other  source,  with  the  exception  of  that  from  a  form  of  "  puerperal 
cystitis "  which  Bumm  has  described.  But  in  this  the  diplococci 
contained  in  the  pus  cells  were  to  be  distinguished  by  the  fact  that 
they  retained  their  color  when  treated  by  Gram's  method.  Owing 


PYOGENIC   BACTERIA.  297 

to  the  difficulty  of  cultivating  this  micrococcus,  and  the  importance, 
under  certain  circumstances,  of  not  making  a  mistake  in  its  diag- 
nosis, these  characters  are  of  exceptional  value. 

Biological  Characters. — Bumm  (1885)  first  succeeded  in  culti- 
vating the  "  gonococcus "  upon  human  blood  serum,  obtained  from 
the  placenta  of  a  recently  delivered  woman.  He  found  that  the  cul- 
tures thrive  best  in  a  moist  atmosphere  at  30°  to  34°  C.  The  growth 
under  the  most  favorable  conditions  is  slow,  and  frequently  no  devel- 
opment occurs  when  pus  containing  numerous  gonococci  is  placed 
upon  blood  serum  in  an  incubating  oven;  or  after  a  slight  multi- 
plication development  ceases  and  the  cocci  undergo  degenerative 
changes  and  quickly  disappear. 

Cultures  upon  the  surface  of  blood  serum  form  a  very  thin,  often 
scarcely  visible  layer,  with  a  smooth,  moist,  shining  surface,  and 
by  reflected  light  a  grayish-yellow  color.  The  growth  at  the  end  of 
twenty-four  hours  may  extend  for  a  distance  of  a  millimetre  along 
the  line  of  inoculation,  but  at  the  end  of  two  or  three  days  no  fur- 
ther development  occurs  and  the  cocci  soon  lose  their  vitality.  This 
micrococcus,  then,  is  aerobic.  Whether  it  may  also  be  a  facultative 
anaerobic  has  not  been  definitely  determined,  but  it  does  not  grow 
along  the  line  of  puncture  when  stick  cultures  are  made  in  blood  se- 
rum. Its  rapid  and  abundant  multiplication  in  gonorrhoeal  infection 
of  mucous  membranes,  and  the  difficulties  attending  its  cultivation 
in  artificial  media,  show  that  the  gonococcus  is  a  strict  parasite. 

Lestikow  and  Loffler,  prior  to  the  publication  of  Bumm's  impor- 
tant monograph,  had  reported  successful  results  in  cultivating  the 
gonococcus  upon  a  mixture  of  blood  serum  and  gelatin.  Bockhart 
has  since  recommended  a  mixture  of  nutrient  agar  (two  parts),  lique- 
fied at  a  temperature  of  50°  C.,  with  blood  serum  (two  to  three  parts) 
at  20°  C.  By  quickly  mixing  with  this  a  little  pus  containing  the 
gonococcus  he  was  able  to  obtain  colonies  upon  plate  cultures,  made 
by  pouring  the  liquid  medium  upon  sterile  glass  plates  in  the  usual 
manner. 

Ghon  and  Schlagenhaufer  in  1893  reported  that  they  obtained 
good  results  by  adding  phosphate  of  soda  to  blood-serum  agar,  made 
according  to  the  method  of  Wertheim — one  part  of  human  blood 
serum  from  the  placenta  to  two  or  three  parts  of  nutrient  agar.  Also 
that  they  were  successful  in  cultivating  the  gonococcus  in  an  acid 
medium  made  by  adding  one  part  of  urine  to  two  of  nutrient  agar 
(two  per  cent).  Turro  (1894)  has  since  published  the  results  of  his 
experiments  relating  to  the  cultivation  of  this  micrococcus  in  acid 
media.  According  to  him  it  grows  in  normal  urine,  either  with  or 
without  the  addition  of  peptone  (one  per  cent) ;  also  in  acid  gelatin, 
prepared  in  the  usual  way  but  without  neutralization  (?). 


298  PYOGENIC   BACTERIA. 

Turro  also  claims  to  have  produced  specific  urethritis  in  dogs  by 
inoculation  with  his  cultures.  Heiman  (1895)  as  a  result  of  an  ex- 
tended experimental  research,  arrives  at  the  conclusion  that  "the 
diplococcus  described  by  Turro  in  connection  with  his  acid  media  is 
not  the  gonococcus."  His  inoculation  experiments  in  dogs,  made 
with  pure  cultures  of  the  gonococcus,  gave  an  entirely  negative  result. 
For  the  cultivation  of  the  gonococcus,  Heiman  recommends  a  medium 
made  from  "chest  serum"  obtained  from  a  patient  suffering  with 
hydrothorax  or  acute  pleurisy.  This  was  found  to  be  superior  to 
placenta  serum,  sheep-blood  serum,  or  peritoneum  serum,  because  of 
the  great  amount  of  serum  albumin  which  it  contains.  Two  per 
cent  of  agar,  one  per  cent  of  peptone,  and  one-half  per  cent  of  sodium 
chloride  were  added  to  the  chest  serum,  and  the  medium  was  sterilized 
by  "fractional  sterilization." 


Fio.  87.— Gonorrhoeal  conjunctivitis,  second  day  of  sickness;  section  through  the  mucous  mem- 
brane of  upper  eyelid;  invasion  of  the  epithelial  layer  by  gonococci.    (Bumm.) 

Schrotter  and  Winkler  (1890)  report  their  success  in  cultivating 
the  gonococcus  upon  albumin  from  the  egg  of  the  pewit — "Kibitz." 
In  the  culture  oven  at  38°  C.  a  thin,  transparent,  whitish  layer  was 
already  visible  at  the  end  of  six  hours  and  rapidly  extended  ;  the 
growth  was  less  abundant  at  the  end  of  three  days,  and  had  entirely 
ceased  by  the  fifth  day.  Attempts  to  cultivate  the  same  microor- 
ganism in  albumin  from  hens'  eggs  gave  a  negative  result. 

Aufuso  (1891)  has  cultivated  the  gonococcus  in  fluid  obtained 
from  the  knee  joint  in  a  case  of  chronic  synovitis,  but  failed  to  culti- 
vate it  in  the  fluid  of  ascites.  A  culture  of  the  twelfth  generation 
made  upon  the  culture  medium  mentioned,  solidified  by  heat,  was 
introduced  into  the  urethra  of  a  healthy  man  and  gave  rise  to  a 
characteristic  attack  of  gonorrhoea. 

Development  does  not  occur  below  25°  or  above  38°  C.  The 
writer  has  shown  that  a  temperature  of  60°  C.  maintained  for  ten 
minutes  destroys  the  infective  virulence  of  gonorrhceal  pus. 

Pathogenesis.—  That  the  gonococcus  is  the  cause  of  the  specific 
inflammation  and  purulent  discharge  characteristic  of  gonorrhoea  is 
now  generally  admitted  upon  the  experimental  evidence  obtained  by 


PYOGENIC   BACTERIA.  299 

Bumm.  Having  succeeded  in  obtaining  it  in  pure  cultures  from 
gonorrhoeal  pus,  he  made  successful  inoculations  in  the  healthy  ure- 
thra in  two  cases— once  with  a  third  culture  and  once  with  one 
which  had  been  transferred  through  twenty  successive  generations. 
In  both  cases  a  typical  gonorrhoea  developed  as  a  result  of  the  inocu- 
lation. 

The  mucous  membranes  in  man  which  are  subject  to  gonorrhoeal 
infection  are  those  of  the  urethra,  the  conjunctiva,  the  cervix  uteri, 
and  the  vagina  in  children — the  vagina  in  adults  is  not  involved. 
Inoculations  of  gonorrhoeal  pus  into  the  vagina  or  conjunctival  sac  of 
the  lower  animals — dogs,  rabbits,  horses,  apes — are  without  result. 

The  very  numerous  researches  which  have  been  made  by  compe- 
tent bacteriologists  show  that  the  gonococcus  is  constantly  present  in 
gonorrhoeal  discharges,  and  in  view  of  the  facts  above  stated  its  etio- 
logical  import  appears  to  be  fully  established.  Bumm  has  studied 
the  development  of  blennorrhoea  neonatorum,  and  has  shown  that 
soon  after  infection  the  presence  of  gonococci  may  be  demonstrated 
in  the  superficial  epithelial  cells  of  the  mucous  membrane  and  be- 
tween them  ;  that  they  soon  penetrate  to  the  deeper  layers,  and  that 
by  the  end  of  forty-eight  hours  the  entire  epithelial  layer  is  invaded 
by  the  diplococci,  which  penetrate  by  way  of  the  connecting  mate- 
rial— "  Kittsubstance  " — between  the  cells.  They  also  multiply  in 
the  superficial  layers  of  connective  tissue  and  give  rise  to  an  inflam- 
matory reaction,  which  is  shown  by  an  abundant  escape  of  leuco- 
cytes from  the  dilated  capillary  network.  The  penetration  of  the 
gonococci  to  the  deeper  layers  of  the  mucous  membrane  of  the  ure- 
thra, and  even  to  the  corpus  cavernosum,  was  observed  by  Bockhart 
in  a  case  studied  by  him  in  which  death  occurred  during  an  acute 
attack  of  gonorrhoea.  But  Bumm  concludes  from  his  researches 
that  this  is  not  usual,  and  that  the  invasion  is  commonly  limited  to 
the  superficial  layers  of  the  mucous  membrane. 

Staphylococcus  pyogenes  aureus  is  not  infrequently  associated 
with  the  gonococcus  in  late  gonorrhoeal  discharges,  and  the  abscesses 
which  occasionally  develop  as  a  complication  of  gonorrhoea,  in  the 
prostate,  the  inguinal  glands,  or  around  the  urethra,  are  probably 
due  to  its  presence,  which  has  been  demonstrated  in  the  pus  from 
such  abscesses  in  a  number  of  cases.  The  same  is  true  of  the  joint 
affections  and  endocarditis  which  sometimes  occur  in  the  course  of 
an  attack  of  gonorrhoea.  Although  some  authors  have  claimed  to 
find  the  gonococcus  in  these  so-called  metastatic  gonorrhoeal  inflam- 
mations, the  evidence  is  not  satisfactory,  and  it  seems  probable  that 
the  Staphylococcus  aureus  is  the  usual  microorganism  concerned  in 
these  affections. 


V. 
BACTERIA  IN  CROUPOUS  PNEUMONIA. 

THE  following  account  of  "  The  Etiology  of  Croupous  Pneumo- 
nia "  is  from  a  paper  read  by  the  writer  at  the  annual  meeting  of  the 
Medical  Society  of  the  State  of  New  York,  at  Albany,  N.  Y.,  Feb- 
ruary 6th,  1889  : 


Quain's  "Dictionary  of  Medicine,"  says: 

servers  that,  like  the  specific  fevers,  it  is  due  to  a  specific  cause.  Pneumonia, 
whilst  differing  from  these  fevers  in  not  being-  contagious,  resembles  them 
in  the  typical  character  of  its  clinical  phenomena  and,  to  a  less  extent,  of  its 
local  lesion.  The  changes  in  the  lung  occurring  in  pneumonia  cannot  be 
induced  by  artificial  injury  of  the  organ,  and  it  must  therefore  be  admitted 
that  there  is  something  special  in  the  inflammatory  process." 

This  "something  special  "has  been  demonstrated  by  recent  researches, 
and  it  is  the  object  of  the  present  paper  to  give  a  historical  account  of  the  de- 
velopment of  our  knowledge  with  reference  to  this  specific  infectious  agent, 
and  of  the  experimental  evidence  upon  which  the  claim  is  founded  that  the 
microorganism  referred  to  bears  an  etiological  relation  to  the  disease  in 
question. 

Evidently,  if  pneumonia  is  a  specific  infectious  disease,  the  microorgan- 
ism which  causes  it  is  widely  distributed,  and  the  development  of  an  attack 
depends  rather  upon  secondary  predisposing  and  exciting  causes  than  upon 
the  accidental  introduction  of  the  specific  agent. 

It  cannot  be  maintained  that  the  disease,  as  a  general  rule,  is  transmitted 
from  individual  to  individual — i.e.,  by  personal  contagion.  Clinical  expe- 
rience is  entirely  opposed  to  this  view,  although  we  have  ample  evidence 
that  it  may  occur  as  an  epidemic  among  individuals  who  are  exposed  to  the 
same  conditions  of  environment — as  in  jails,  barracks,  etc.  Thus  at  Chris- 
tiania,  Sweden,  an  epidemic  of  pneumonia  occurred  in  1847  in  the  prison, 
during  which  sixty-nine  of  the  prisoners  were  attacked.  And  again  in  1866 
and  1867,  during  a  period  of  six  months  (December,  1866,  to  May,  1867),  a 
similar  epidemic  was  observed  in  the  same  prison — sixty-two  cases.  Other 
prison  epidemics  recorded  are  those  at  Frankfort  in  1875  (seventy -five  cases) 
and  in  1876  (ninety-eight  cases) ;  at  Maringen  in  1875  (eighty-three  cases) 
and  in  1878  (fifty-eight  cases) ;  at  the  prison  of  DAnsberg  in  1880  (one  hun- 
dred and  sixty-one  cases,  with  forty-six  deaths,  in  a  period  of  five  months). 

Ajrain,  we  have  numerous  records  of  village  epidemics  and  of  epidemics 
confined  to  single  houses.  In  outbreaks  of  this  character,  as  in  epidemics 
of  typhoid  fever,  of  cholera,  and  of  yellow  fever,  there  is  a  succession  of 
cases  occurring  at  different  intervals,  but  it  does  not  follow  that  these  cases 
bear  any  direct  relation  to  each  other.  On  the  contrary,  everything  indi- 
cates that,  as  in  the  diseases  mentioned,  in  the  presence  of  the  infectious 


BACTERIA   IN   CROUPOUS   PNEUMONIA.  301 

agent  common  predisposing  causes  relating  to  the  environment,  acting  upon 
persons  having  various  degrees  of  resisting  power,  induce  attacks  at  various 
intervals  ;  or  it  may  be  that  in  the  presence  of  the  specific  cause  and  predis- 
posing influences  an  exciting  cause,  such  as  exposure  to  cold,  alcoholic  ex- 
cess, etc.,  is  the  immediate  factor  in  the  development  of  an  attack. 

Without  stopping  to  discuss  further  the  facts  relating  to  the  epidemic 
prevalence  of  the  disease  under  consideration,  I  call  attention  to  the  well- 
established  fact  that  pneumonia  prevails  over  a  wide  area  of  the  inhabited 
surface  of  the  earth,  and  that  by  far  the  larger  number  of  cases  occur  inde- 
pendently of  any  recognized  connection  with  previous  cases,  and  often  un- 
der circumstances  in  which  such  connection  can  be  very  positively  excluded. 
And,  on  the  other  hand,  the  direct  transmission  of  the  disease  by  the  sick  to 
those  most  closely  associated  with  them,  as  nurses,  etc.,  if  it  occurs  at  all,  is 
evidently  a  rare  exception  to  the  general  rule. 

We  must  then  conclude,  as  stated  at  the  outset,  that  if  pneumonia  is  a 
specific  infectious  disease  the  microorganism  which  causes  it  is  widely  dis- 
tributed. As  a  matter  of  fact,  the  pathogenic  micrococcus  which,  from  the 
evidence  now  at  hand,  appears  to  be  the  specific  etiological  agent  in  acute 
pneumonia  has  been  found  in  the  buccal  secretions  of  healthy  individuals 
in  various  parts  of  the  world — in  America,  in  France,  in  Italy,  and  in  Ger- 
many, and  no  doubt  more  extended  researches  will  show  that  it  is  extremely 
common. 

This  statement  may  appear  at  the  outset  to  make  the  view  that  the  micro- 
coccus  in  question  is  the  cause  of  croupous  pneumonia  quite  untenable. 
For,  it  may  be  asked,  how  is  it  that  the  individuals  who  have  this  microor- 
ganism in  their  buccal  secretions  escape  an  attack  of  pneumonia  ?  In  the 
present  state  of  our  knowledge  this  question  no  longer  presents  any  serious 
difficulties.  We  know,  for  example,  that  the  pus  organisms— Staphylococcus 
pyogenes  aureus,  albus,  and  citreus — are  very  frequently  found  in  the  buc- 
cal secretions  and  on  the  surface  of  the  body  of  healthy  individuals,  and 
that,  although  these  micrococci  are  recognized  as  the  cause  of  furuncles  and 
of  all  sorts  of  acute  abscesses,  they  only  give  rise  to  the  formation  of  such 
abscesses  under  certain  special  conditions  relating  to  the  general  health  of 
the  individual,  or  to  a  traumatism  by  which  their  introduction  to  vulnerable 
parts  is  effected.  Again,  the  tetanus  bacillus  is  a  widely  distributed  micro- 
organism which  has  been  found  in  the  earth,  and  especially  in  rich  loam,  in 
various  parts  of  the  globe.  But  the  hands  of  farmers  and  gardeners  are  con- 
stantly soiled  with  such  earth  without  their  contracting  tetanus.  In  this 
instance  it  has  long  been  recognized  that  a  traumatism  is  an  essential  factor 
in  the  chain  of  events  which  leads  to  the  development  of  tetanus,  and  now 
we  believe,  on  satisfactory  experimental  evidence,  that  it  is  not  the  trauma- 
tism per  se,  or  the  injury  to  the  nerves,  or  exposure  to  cold,  which  in  certain 
cases  gives  rise  to  this  infectious  malady,  but  that  the  result  depends  upon 
the  introduction  of  a  specific  infectious  agent  at  the  time  the  wound  was  re- 
ceived or  subsequently — the  tetanus  bacillus  of  Nicolaier. 

In  the  case  of  the  tubercle  bacillus,  also,  it  is  extremely  probable,  in  the 
light  of  our  present  knowledge,  that  this  bacillus,  in  a  living  condition,  not 
infrequently  finds  a  lodgment  in  the  mouth,  upon  the  Schneiderian  mucous 
membrane,  or  in  the  larger  bronchial  tubes  of  most  individuals  who  live  in 
populous  communities.  Here  also  the  infectious  agent  is  only  one  factor, 
although  an  essential  one,  in  the  production  of  the  infectious  disease.  It 
must  be  introduced  to  the  vulnerable  location,  and  must  find  a  favorable 
nidus  in  the  tissues  invaded.  We  have  good  reason  to  believe  that  in  this, 
as  well  as  in  other  infectious  diseases,  there  are  wide  differences,  inhe- 
rited or  acquired,  in  the  susceptibility  of  the  tissues  to  invasion  when  the 
infectious  agent  has  been  introduced  to  a  favorable  location. 

In  a  paper  read  before  the  Pathological  Society  of  Philadelphia  in  April, 
1885,  in  discussing  the  relation  of  my  Micrococcus  Pasteuri  to  croupous 
pneumonia,  I  say:  "It  seems  extremely  probable  that  this  micrococcus  is 
concerned  in  the  etiology  of  croupous  pneumonia,  and  that  the  infectious 


302  BACTERIA  IN   CROUPOUS  PNEUMONIA. 

nature  of  this  disease  is  due  to  its  presence  in  the  fibrinous  exudate  into  the 
pulmonary  alveoli. 

"But  this  cannot  be  considered  as  definitely  established  by  the  experi- 
ments which  have  thus  far  been  made  upon  the  lower  animals.  The  con- 
stant '  presence  of  this  micrococcus  in  the  buccal  secretions  of  healthy  per- 
sons indicates  that  some  other  factor  is  required  for  the  development  of  an 
attack  of  pneumonia;  and  it  seems  probable  that  this  other  factor  acts  by  re- 
ducing the  vital  resisting  power  of  the  pulmonary  tissues,  and  thus  making 
them  vulnerable  to  the  attacks  of  the  microbe.  This  supposition  enables  us 
to  account  for  the  development  of  the  numerous  cases  of  pneumonia  which 
cannot  be  traced  to  infection  from  without.  The  germ  being  always  pre- 
sent, auto-infection  is  liable  to  occur  when,  from  alcoholism,  sewer-gas 
poisoning,  crowd  poisoning,  or  any  other  depressing  agency,  the  vitality  of 
the  tissues  is  reduced  below  the  resisting  point.  We  may  suppose,  also,  that 
a  reflex  vaso- motor  paralysis,  affecting  a  single  lobe  of  the  lung,  for  exam- 
ple, and  induced  by  exposure  to  cold,  may  so  reduce  the  resisting  power  of 
the  pulmonary  tissue  as  to  permit  this  micrococcus  to  produce  its  character- 
istic effects. 

"  Again,  we  may  suppose  that  a  person  whose  vital  resisting  power  is 
reduced  by  any  of  the  causes  mentioned  may  be  attacked  by  pneumonia 
from  external  infection  with  material  containing  a  pathogenic  variety  of 
this  micrococcus  having  a  potency,  permanent  or  acquired,  greater  than  that 
possessed  by  the  same  organism  in  normal  buccal  secretions." 

This  is  the  theory  by  which  I  have  attempted  to  explain  the  etiological 
role  of  this  micrococcus  in  croupous  pneumonia.  Let  us  now  consider  the 
principal  facts  which  have  led  to  a  belief  in  its  causal  relation  to  this  disease. 

Friedlander,  in  1882,  observed,  in  eight  fatal  cases  of  pneumonia  in  which 
he  made  autopsies,  microorganisms,  having  an  oval  form,  in  the  exudate  into 
the  pulmonary  alveoli ;  they  were  in  pairs  or  in  short  chains.  Without  af- 
firming that  this  microorganism  is  the  cause  of  pneumonia,  Friedlander 
seems  to  have  considered  it  extremely  probable  that  it  bore  an  etiological  re- 
lation to  this  disease. 

During  the  same  year  Leyden  and  Gunther  announced  at  a  meeting  of 
the  Medical  Society  of  Berlin  (November  20th,  1882)  that  they  had  found  the 
same  micrococcus  in  the  fibrinous  exudate  of  pneumonia,  obtained  through 
the  thoracic  walls  by  means  of  a  Pravaz  syringe.  At  the  same  time  Gunther 
stated  that  the  elliptical  cocci,  in  specimens  stained  with  gentian  violet,  were 
surrounded  with  a  colorless  capsule. 

The  following  year  Matruy  published  his  observations.  In  sixteen  cases 
he  had  found  an  elongated  coccus  in  the  fibrinous  exudate  of  pneumonia,  and 
always  having  a  very  transparent  capsule.  He  had  also  encountered  the 
same  microorganism  in  the  sputa  of  patients  with  other  diseases,  but  not  so 
abundantly  as  in  pneumonia. 

On  November  19th,  1883,  Friedlander  communicated  to  the  Medical  Soci- 
ety of  Berlin  the  results  of  his  culture  and  inoculation  experiments.  His 
"pneumococcus"  was  characterized  by  the  presence  of  a  capsule  which,  as 
he  says,  "  always  takes  the  form  of  the  microorganism;  if  this  is  round  the 
capsule  is  round;  if  it  is  elliptical  the  capsule  is  an  ellipse."  This  capsule, 
however,  was  only  found  in  preparations  made  from  the  blood  of  an  inocu- 
lated animal  or  from  the  fibrinous  exudate  into  the  alveoli ;  in  cultures  it 
was  no  longer  seen.  The  cultures  in  flesh-peptone  gelatin  presented  a  nail- 
shaped  growth  which  was  believed  to  be  characteristic.  Growth  was  rapid 
in  a  variety  of  culture  media  at  the  ordinary  room  temperature  (65°  to 
75°  F.),  and  in  a  gelatin  culture  medium  no  liquefaction  occurred. 

The  following  results  were  obtained  by  Friedlander  in  his  inoculation  ex- 
periments: In  one  series  of  experiments  the  "pneumococci,"  mixed  with 
distilled  water,  were  injected  through  the  thoracic  walls  into  the  lungs. 
Nine  rabbits  inoculated  in  this  way  gave  an  entirely  negative  result.  Six 

1 1  should  have  said  frequent  instead  of  "  constant  presence." 


BACTERIA  IN   CROUPOUS  PNEUMONIA.  303 

out  of  eleven  guinea-pigs  are  said  to  have  succumbed  and  to  have  presented 
the  lesions  of  pneumonia.  All  of  the  mice  injected  died  within  twenty -four 
hours,  and  at  the  autopsy  the  lungs  were  found  to  be  congested  and  to  pre- 
sent foci  of  red  hepatization.  In  a  second  series  of  experiments  upon  mice 
they  were  made  to  inhale  a  spray  containing  the  pneumococci  in  suspension. 
Several  of  these  animals  died  and  are  said  to  have  presented  a  typical  pneu- 
monia. The  "  pneumococcus, "  surrounded  by  its  characteristic  capsule,  was 
found  in  the  lungs,  the  spleen,  the  blood,  and  the  liquid  contained  in  the 
pleural  cavity. 

Upon  this  evidence  Friedlander's  "pneumococcus,"  which  is  now  usually 
described  as  a  bacillus,  was  very  generally  accepted  as  the  specific  cause  of 
flbrinous  pneumonia,  and  cultures  were  distributed  throughout  the  labora- 
tories of  Europe  bearing-  the  label,  "  Pneumococcus  of  Friedlander."  For 
some  time  after  the  publication  of  Friedlander's  paper  all  observations  made 
with  reference  to  the  presence  of  oval  cocci  or  of  encapsulated  cocci  in  the 
fibrinous  exudate  of  pneumonia  were  supposed  to  confirm  his  discovery. 
But  now  we  know  that  there  is  another  oval  coccus  which  is  far  more  fre- 
quently present  in  the  exudate  of  acute  pneumonia,  which  also  presents  the 
appearance  of  being  surrounded  by  a  transparent  capsule — less  pronounced, 
however,  than  that  of  Friedlander's  bacillus — but  which  is  entirely  distinct 
from  that  of  Friedlander  and  is  probably  the  true  pneumococcus.  I  shall 
give  the  distinctive  characters  of  this  microorganism  later. 

At  the  same  time  that  Friedlander  was  pursuing  his  researches  in  Berlin, 
Talamon,  a  French  physician,  was  engaged  in  similar  researches  in  the  lab- 
oratory of  the  Hotel-Dieu.  His  results  were  communicated  to  the  Anato- 
mical Society  of  Paris  on  November  30th,  1883,  a  few  days  after  Friedlan- 
der's communication  to  the  Medical  Society  of  Berlin  (Germain  See). 

"  Talamon  did  not  describe  his  microbe  as  having  a  capsule;  according 
to  him,  the  pneumonia-coccus  is  characterized  by  its  form.  When  seen  in 
the  fibrinous  exudate  the  microbe  has  an  elliptical  form,  like  a  grain  of 
wheat.  Cultivated  in  a  liquid  medium — an  alkaline  solution  of  extract  of 
beef — it  is  elongated  and  attenuated,  and  presents  the  appearance  of  a  grain  of 
barley.  On  account  of  this  appearance  Talamon  has  proposed  to  call  it  the 
lanceolate  coccus.  This  organism  is  encountered  in  the  pneumonic  exudate 
obtained  after  death,  or  drawn  during  life  by  means  of  a  Pravaz  syringe 
from  the  hepatized  portions  of  the  lung.  Once  only,  out  of  twenty-five 
cases,  it  was  found  in  the  blood  of  a  patient  at  the  moment  of  death." 

Talamon's  inoculation  experiments  in  dogs  and  guinea-pigs  gave  a  nega- 
tive result,  but  out  of  twenty  rabbits  injected  through  the  walls  of  the 
thorax  into  the  lungs  eight  showed  the  characteristic  lesions  of  fibrinous 
pneumonia.  Prof.  See  says,  with  reference  to  the  evidence  in  the  case  of 
these  rabbits  as  compared  with  that  obtained  by  Friedlander  in  his  mice: 
44  The  rather  brief  description  of  the  lesions  obtained  by  Friedlander  in  the 
mice  inoculated  by  him  leaves  some  doubt  in  the  mind;  for  the  presence  of 
foci  (noyaux)  of  induration  in  congested  lungs  is  not  sufficient  to  character- 
ize fibrinous  pneumonia.  But  the  lungs  of  the  rabbits  presented  by  Tala- 
mon to  the  Anatomical  Society  in  support  of  his  communication  leave  no 
room  for  discussion.  As  he  observed,  it  was  not  at  all  a  question  of  foci  of 
congestion,  or  of  broncho-pneumonia,  such  as  one  observes  habitually  in 
rabbits  which  die  of  septicaemia,  but  a  veritable  lobar  fibrinous  pneumonia 
with  pleurisy  and  pericarditis  of  the  same  nature.  The  naked-eye  examina- 
tion, as  well  as  the  microscope,  showed  no  difference  in  the  lesions  produced 
in  the  rabbit  and  the  pneumonia  of  man." 

On  another  page  Prof.  See  says  :  4 '  Afanassiew  repeated  in  the  laboratory 
of  Prof.  Cornil  the  experiments  of  Friedlander  and  of  Talamon  ;  by  the  cul- 
ture in  peptonized  gelatin  of  the  pneumonic  exudate  taken  from  the  cadaver 
he  obtained  two  species  of  organisms,  round  micrococci  of  large  and  small 
dimensions,  and  oval  cocci  which  corresponded  to  the  microbes  described  by 
the  two  authors  "  (Friedlander  and  Talamon)  '4  whose  researches  we  have 
just  reviewed."  This  quotation  indicates  that  Prof.  See  did  not  question  the 


304  BACTERIA  IN  CROUPOUS  PNEUMONIA. 

identity  of  the  oval  or  "  lanceolate  "  coccus  found  by  Talamon  in  pneumonic 
exudate,  and  which  in  his  experiments  produced  typical  pneumonia  in  rab- 
bits, and  the  so-called  "  pneumococcus  "  of  Friedlander,  which,  according 
to  his  account,  gave  a  negative  result  when  injected  into  rabbits,  but  caused 
pneumonia  in  mice  when  injected  directly  into  the  lungs.  Prof.  See  was 
not  alone  in  making  this  inference,  which  has  turned  out  to  be  a  mistaken 
one.  The  identity  of  the  oval  cocci,  which  had  now  been  seen  in  the  pul- 
monary exudate  by  numerous  observers,  with  the  microorganism  which 
Friedlander  had  isolated  and  cultivated  from  material  obtained  post  mortem 
from  hepatized  lungs,  was  generally  admitted ;  and  all  of  the  observations 
relating  to  the  presence  of  oval  cocci,  having  a  more  or  less  distinct  capsule, 
in  the  exudate  of  fibrinpus  pneumonia,  were  supposed  to  give  support  to  the 
alleged  discovery  of  Friedlander.  Now  we  know  that  the  oval  coccus  most 
frequently  found  in  such  material  is  not  that  of  Friedlantier,  but  that  it  is 
identical  with  a  coccus  first  observed  by  the  writer  in  September,  1880,  in 
the  blood  of  rabbits  injected  with  his  own  saliva  and  subsequently  (1885) 
named  by  him  Micrococcus  Pasteuri. 

This  was,  without  doubt,  the  coccus  which  produced  pneumonia  in  Tala- 
mon's  experiments  upon  rabbits ;  and  we  must  give  him  the  credit  of  having 
first  experimentally  aempnstrated  the  fact  that  fibrinous  pneumonia  may  be 
induced  by  the  introduction  of  this  microorganism  into  the  parenchyma  of 
the  lung  in  these  animals. 

Salvioli,  whose  experiments  were  also  made  in  1884,  had  a  uniformly 
fatal  result  from  the  injection  of  pneumonic  sputum  into  rabbits  (four).  He 
also  observed  the  oval  coccus  in  the  material  injected,  and  in  the  blood  of 
the  animals  which  succumbed  to  his  injections,  but  did  not  recognize  the 
identity  of  this  coccus  with  that  which  my  own  experiments  and  those  of 
Pasteur,  Vulpian,  and  others  had  shown  to  be  present  in  normal  human 
saliva  and  to  induce  a  fatal  form  of  septicaemia  in  rabbits.  On  the  other 
hand,  he  also  appears  to  have  taken  it  for  granted  that  the  oval  micrococcus 
encountered  by  him,  and  which,  under  certain  circumstances,  was  sur- 
rounded by  a  transparent  capsule,  was  the  '*  pneumococcus  "  of  Friedlander. 
Klein  appears  to  have  made  the  same  mistaken  inference.  This  is  shown 
by  the  following  quotation  from  his  paper  published  in  1885: 

44  In  seeking  to  ascertain  what  might  be  the  relation  between  the  so-called 
pneumococci  and  croupous  pneumonia,  I  have  made  extensive  examination 
of  the  lungs  and  blood  of  persons  dead  of  the  disease,  and  also  of  the  sputum 
of  living  patients  at  various  stages  of  their  illness.  ...  In  some  of  the  air 
vesicles,  tnough  few  and  far  between,  there  were  present  undoubtedly  the 
capsulated  cocci  spoken  of  by  Friedlander  and  others  as  pneumococci.  .  .  . 
As  regards  the  living  patients,  if  we  examine  tvpical  sputum  of  croupous 
pneumonia  we  find,  besides  numerous  red  blood  discs  and  white  blood  cor- 
puscles, also  a  few  epithelial  cells,  and  in  the  general  gelatinous  matrix 
numbers  of  microorganisms,  chiefly  belonging  to  the  species  micrococci.  .  .  . 

4 'These  are,  as  far  as  size  and  arrangement  go,  of  two  principal  types: 
(a)  Oval  micrococci  about  0.001  millimetre  in  length,  occurring  isolated,  but 
more  commonly  as  dumbbells  and  slightly  curved  chains  of  four,  six,  and 
even  eight  elements.  .  .  .  But  in  all  tnese  micrococci  the  elements  are  dis- 
tinctly surrounded  by  a  hyaline  zone  which,  in  stained  preparations,  can  be 
made  out  as  an  unstained  halo,  though  in  some  stained  specimens  it  as- 
sumes a  tint  that  is  fainter  than  that  of  the  micrococcus  itself;  this  corre- 
sponds to  the  capsule  of  Friedlander,  and  for  this  reason  he  called  them 
capsule  micrococci." 

In  a  footnote  to  the  paper  from  which  I  have  quoted  Klein  says: 

44  While  this  paper  is  passing  through  the  press  I  receive  from  Dr.  Stern- 
berg,  of  Baltimore,  a  paper  in  which  he  conclusively  proves  that  the  mi- 
crococci of  human  saliva,  which  produce  in  some  instances  septicaemia  on 
inoculation  into  rabbits,  are  identical  with  the  pneumococci  of  Friedlander, 
Salvioli,  and  others." 

My  own  experiments  with  pneumonic  sputum  were  made  in  January, 


BACTERIA  IN  CROUPOUS  PNEUMONIA.  395 

1885,  and  led  me  to  the  identification  of  the  oval  coccus  found  in  this  ma- 
terial with  the  coccus  found  in  my  own  saliva  (by  inoculations  into  rabbits) 
in  September,  1880,  and  subsequently  studied  by  me  in  an  extended  series 
of  experiments  made  during  the  following  years,  1880-84. 

But,  at  the  same  time,  I  fell  into  the  error  of  inference,  previously  made 
by  Prof.  See,  by  Salvioli,  and  others,  and  assumed  that  the  "pneumo- 
coccus  "  which  Friedlander  had  obtained  from  the  same  source  was  the  same, 
although  I  found  it  difficult  to  reconcile  the  experimental  data,  inasmuch 
as  he  had  obtained  uniformly  negative  results  in  his  inoculations  into 
rabbits.  To  explain  this  discrepancy  I  suggested  that  Friedlander's  pneu- 
mococcus  was  probably  a  variety  having  a  different  degree  of  pathogenic 
power. 

This  supposition  seemed  to  find  support  in  the  fact,  which  I  had  previ- 
ously observed,  that  my  Micrococcus  Pasteuri  became  attenuated,  as  to  its 
pathogenic  power,  when  the  cultures  were  kept  for  some  time ;  and  that 
there  seemed,  from  the  experimental  evidence  before  me,  to  be  different 


recognized  my  mistake  and  hastened  to  correct  the  error.1 

For  a  detailed  account  of  my  experiments  with  pneumonic  exudate  I 
must  refer  to  my  paper  published  in  the  ' '  Transactions  of  the  Pathological 
Society  of  Philadelphia"  (vol.  xii.)  and  in  the  American  Journal  of  the 
Medical  Sciences  (July,  1885). 

With  reference  to  my  conclusion  that  the  oval  coccus  of  Talamon  and 
of  Salvioli  was  identical  with  my  Micrococcus  Pasteuri,  I  may  say  that 
this  conclusion  has  been  sustained  by  the  subsequent  investigations  of 
Frankel,  Weichselbaum,  Bordoni-Uffreduzzi,  Netter,  Gameleia,  and  others. 

Frankel's  first  paper  relating  to  the  presence  of  this  microorganism  in 
pneumonic  exudate  was  published  in  1885. 

Having  ascertained  that  his  own  saliva  contained  this  oval  micrococcus, 
he  was  induced  to  make  an  extended  and  interesting  series  of  experiments 
which  led  him  to  the  conclusion  that  this  microorganism  is  far  more  con- 
stantly present  in.  the  exudate  of  fibrinous  pneumonia  than  is  the  so-called 
" pneumococcus "  of  Friedlander.  He  says: 

"Finally,  as  regards  the  relative  frequency  of  the  two  hitherto  investi- 
gated microorganisms  in  cases  of  pneumonia,  no  positive  statement  can  yet 
be  made.  Nevertheless  I  am  inclined  to  regard  the  lancet-shaped  pneu- 
mococcus, which  is  identical  with  the  microbe  of  sputum  septicaemia,  as  the 
more  frequent,  and  the  usual  infectious  agent  of  pneumonia,  on  the  ground 
that  this  organism  is  so  much  more  frequently  found  in  the  sputum  of  pneu- 
monic patients  than  in  that  of  healthy  individuals.  This  conclusion  is 
further  supported  by  the  fact  that  it  has  not  hitherto  been  possible  to  isolate, 
directly  from  the  rusty  sputum,  Friedlander's  bacillus." 

The  extended  researches  of  Weichselbaum,  published  in  1886,  give  strong 
support  to  the  view  that  this  coccus  is  the  usual  infectious  a,gent  in  croupous 
pneumonia.  He  examined,  in  all,  the  exudate  in  one  hundred  and  twenty- 
nine  cases  of  pneumonia. 

In  ninety-four  of  these  cases  the  micrococcus  in  question,  called  by 
Weichselbaum  "  diplococcus  pneumonias, "  was  obtained  (fifty-four  times  in 
cultures);  in  twenty-one  cases  he  obtained  a  streptococcus,  and  in  nine  only 
was  the  bacillus  of  Friedlander  encountered. 

Wolf,  whose  studies  were  made  in  Weichselbaum's  laboratory,  reported 
the  result  of  his  researches  in  1887.  He  found  the  "diplpcoccus  pneumoniae" 
in  sixty-six  out  of  seventy  cases  of  croupous  pneumonia  examined,  and  the 
"  pneumococcus  of  Friedlander  "  in  three  cases. 

Netter,  whose  paper  was  published  in  November,  1887,  found  Micrococcus 

1  See  my  paper  published  in  the  American  Journal  of    the  Medical   Sciences 
for  July,  1886. 
20 


300  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

Pasteuri  in  seventy-five  per  cent  of  his  cases  of  pneumonia,  and  in  the  sputum 
of  convalescents  from  this  disease  its  presence  was  verified  in  sixty  per  cent 
of  the  cases  by  inoculation  experiments  in  rabbits.  He  makes  the  interest- 
ing observation  that  the  sputum  of  recent  convalescents  is  less  virulent  for 
rabbits  than  that  collected  at  a  later  period. 

Gameleia,  who  has  recently  published  in  the  Annales  of  the  Pasteur 
Institute  an  important  paper  upon  the  etiology  of  fibrinous  pneumonia,  veri- 
fied the  presence  of  Micrococcus  Pasteuri  in  twelve  fatal  cases  in  which  he 
collected  material  post  mortem.  He  states  that  in  a  series  of  forty  con- 
secutive cases  Dr.  Goldenberg,  whose  experiments  were  made  in  his  laboratory, 
found  this  micrococcus  in  everv  case  by  inoculation  experiments  in  rabbits  or 
in  mice.  According  to  Gameleia,  inoculations  in  mice  are  more  reliable  than 
those  made  in  rabbits,  as  the  mouse  is  the  more  susceptible  animal.  He  says: 
"The  author,  Weichselbaum,  who  has  made  the  most  extended  research 
upon  the  etiology  of  pneumonia,  used  in  his  researches  the  method  of  culti- 
vation upon  gelatin.  We  must  adopt  the  opinion  of  Baumgarten,  who  does 
not  accord  any  decided  value  to  the  negative  results  of  Weichselbaum  with 
reference  to  the  constant  presence  of  Streptococcus  Paste.uri.  Netter,  who 
adopted  the  method  of  inoculating  the  pneumonic  sputum  into  rabbits,  and 
who  only  found  the  microbe  of  Pasteur  in  seventy-five  per  cent  of  his  cases, 
was  wrong,  in  our  opinion,  in  making  use  of  an  animal  which  is  too  resist- 
ant to  determine  the  presence  of  small  quantities  of  virus.  This  opinion  is 
confirmed  by  the  fact  that  Netter  rendered  some  rabbits  refractory  by  his 
inoculations  with  material  in  which  he  had  not  found  the  specific 
microbe. 

"  En  rteumt,  taking  our  stand  upon  the  positive  results  which  we  have 
always  obtained,  as  well  as  upon  the  superiority  of  the  method  of  research 
(inoculations  in  mice)  which  wre  have  adopted,  we  believe  ourselves  au- 
thorized to  conclude  that  fibrinous  pneumonia  is  always  dependent  upon 
the  microbe  of  Pasteur." 

Frankel,  Weichselbaum,  and  other  recent  authors,  while  maintaining 
that  Micrococcus  Pasteuri  is  the  most  frequent  etiological  agent  in  the  pro- 
duction of  pneumonia,  have  been  disposed  to  admit  that  in  a  certain  propor- 
tion of  the  cases  the  bacillus  of  Friedlander,  and  possibly  other  microorgan- 
isms, may  bear  the  same  relation  to  the  pneumonic  process.  Gameleia,  on 
the  other  hand,  believes  that  the  bacillus  of  Friedlander  is  a  simple  sapro- 
phyte, the  occasional  presence  of  which  in  pneumonic  exudate  is  without 
etiological  import.  He  remarks  as  follows : 

"  We  may  be  brief  as  regards  the  second  objection  made  against  the  etio- 
logical unity  of  fibrinous  pneumonia,  viz.,  with  reference  to  the  etiologicai 
rights  of  the  microbe  of  Friedlander.  This  microbe  is  found  in  normal  sali- 
va, it  is  a  true  saprophyte,  and  may  at  times  invade  the  diseased  or  dead 
lung.  Weichselbaum  only  found  it  in  seven  per  cent  of  his  cases,  and  al- 
most always  associated  with  other  microbes,  for  he  only  encountered  it  pure 
in  three  cases.  As  to  the  researches  of  the  authors  who  preceded  Frankel, 
it  is  sure  that  the  microbe  which  they  often  found  in  sections  of  diseased 
lungs,  and  which  they  called  the  microbe  of  Friedlander,  was  in  fact  the  mi- 
crobe of  Pasteur,  since  it  was  colored  by  the  method  of  Gram,  which  decol- 
orizes the  bacillus  of  Friedlander.  Many  of  the  positive  results,  then, 
which  have  been  reported  relative  to  the  last-mentioned  microorganism, 
oufHht  to  be  put  to  the  account  of  the  other." 

This  opinion  the  present  writer  has  entertained  since  his  researches  made 
in  1885. 

The  experimrntal  evidence  oflVred  l>y  (Jameleia  in  favor  of  the  etiologi- 
cal role  of  this  micrococcus  is  most  important. 

It  will  be  remembered  that  Talamon  produced  typical  pneumonia  in 
eight  rabbits,  in  1883,  by  inoculating  them  through  the  thoracic  walls  with 
pneumonic  exudate.  Gameleia  says : 

4 'The  number  of  ray  rabbits  iu  which  pneumonia  was  induced  is  about 
two  hundred," 


BACTERIA   IN   CROUPOUS   PNEUMONIA.  307 

The  writer  found  in  his  experiments,  made  in  1881,  that  in  making-  a 
series  of  inoculations  in  rabbits  the  virus  increased  in  virulence,  and  that, 
on  the  other  hand,  the  micrococcus  lost  its  virulence  when  the  cultures 
were  kept  for  some  time.  This  fact  has  been  verified  by  the  subsequent  re- 
searches of  Frankel  and  of  Gameleia.  The  last-named  author  has  shown  that 
to  induce  pneumonia  in  very  susceptible  animals,  like  the  rabbit,  an  attenu- 
ated variety  of  the  microbe  should  be  injected,  for  the  most  virulent  cul- 
tures quickly  cause  death  by  septicaemia.  As  he  expresses  it :  "Animals 
which  are  too  susceptible,  like  the  rabbit  and  the  mouse,  do  not  have  pneu- 
monia, because  they  do  not  offer  a  local  reaction ;  the  virus  is  generalized  in 
their  bodies  and  they  die  of  an  acute  septicaemia  " 

On  the  other  hand,  Gameleia  has  shown  that  "  animals  which  are  but 
little  susceptible  to  the  pneumonic  virus  offer  a  local  resistance  which  gives 
rise  to  very  pronounced  reactionary  phenomena  (extended  fibrino-granular 
oedema),  and  consequently  they  present,  as  a  result  of  intrapulmonary  infec- 
tion, a  typical  fibrinous  pneumonia.  Such  animals  are  the  dog  and  the 
sheep." 

In  his  experiments  upon  these  animals  Gameleia  obtained  the  following 
results: 

The  sheep  was  found  to  survive  subcutaneous  inoculations,  unless  very 
large  doses  (five  cubic  centimetres)  of  the  most  potent  virus  were  ad- 
ministered. But  intrapulmonary  inoculation  was  always  followed  by  typi- 
cal fibrinous  pneumonia,  which  in  the  majority  of  cases  proved  fatal. 

The  microbe  was  rarely  found  in  the  blood,  and  successive  inoculations 
from  one  sheep  to  another  were  not  successful.  Death  occurred,  after  an 
intrapulmonary  inoculation,  on  the  third,  fourth,  or  fifth  day.  The  pneu- 
monia produced  was  lobar,  and  was  attended  with  an  extensive  fibrinous 
exudation  in  which  the  coccus  was  found  in  great  abundance.  In  all,  fifty 
sheep  were  experimented  upon. 

The  writer  found  in  his  experiments,  made  in  1881,  that  the  dog  resists 
inoculations  with  this  coccus.  Gameleia  also  obtained  negative  results  when 
moderate  doses  were  injected  beneath  the  skin,  but  states  that  ' '  intrathoracic 
infection  always  causes  a  frank,  fibrinous  pneumonia  which  is  rarely  fatal ; 
recovery  usually  occurs  in  from  ten  to  fifteen  days,  after  the  animal  has 
passed  through  all  the  stages  of  red  and  gray  hepatization  which  character- 
izes this  affection  in  man."  Twelve  dogs  were  experimented  upon. 

This  micrococcus,  then,  which  in  very  susceptible  animals  (mouse,  rabbit) 
invades  the  blood  and  quickly  causes  aeath  by  septicaemia,  when  injected 
through  the  thoracic  walls  in  less  susceptible  animals  (dog,  sheep),  or  in  an 
attenuated  form  in  the  rabbit,  gives  rise  to  the  local  lesions  which  character- 
ize fibrinous  pneumonia. 

Man  comes  in  the  category  of  slightly  susceptible  animals,  as  is  shown 
by  the  comparatively  small  mortality  from  pneumonia,  and  the  fact  that  the 
micrococcus  found  in  the  exudate  into  the  pulmonary  alveoli  does  not  invade 
the  blood,  unless  in  rare  instances.  We  may  therefore  agree  with  Gameleia 
in  the  following  statement : 

"It  is  clear  that  the  results  obtained  in  the  dog  and  the  sheep,  animals 
which  have  but  a  slight  susceptibility,  are  most  applicable  to  human  patho- 
logy." 

In  my  paper  read  before  the  Pathological  Society  of  Philadelphia  in 
April,  1885,  from  which  I  have  already  quoted,  I  say:  "  It  seems  extremely 
probable  that  this  micrococcus  is  concerned  in  the  etiology  of  croupous  pneu- 
monia. .  .  .  But  this  cannot  be  considered  as  definitely  established  by  the 
experiments  which  have  thus  far  been  made  upon  the  lower  animals.1' 

The  experiments  of  Gameleia  go  far  toward  settling  this  question  in  a 
definite  manner,  and,  considered  in  connection  with  those  of  Talamon  and 
Salvioli,  and  the  extended  researches  of  Frankel,  Weichselbaum,  and  Netter, 
leave  but  little  doubt  that  this  is  the  true  infectious  agent  in  acute  lobar 
pneumonia. 


BACTERIA  IN  CROUPOUS   PNEUMONIA. 


7.       BACILLUS   OF   FRIEDLANDER. 

Synonyms. — Pneumococcus  (Friedlander) ;  Bacillus  pneumoniae 
(Flugge). 

Obtained  by  Friedlander  and  Frobenius  in  pure  cultures  (1883) 
from  the  exudate  into  the  pulmonary  alveoli  in  cases  of  croup- 
ous  pneumonia.  Subsequent  researches  show  that  it  is  only  present 
in  a  small  proportion  of  the  cases — nine  times  in  one  hundred  and 
twenty-nine  cases  examined  by  Weichselbaum,  three  times  in  seventy 
cases  examined  by  Wolf. 

Morphology. — Short  rods  with  rounded  ends,  often  so  short  as 

to  resemble  micrococci,  especially  in  very 

(^m         recent  cultures  ;  commonly  united  in  pairs 

°&  ^W  ^@(§\/^     or  chains  of  four,  and  under  certain  cir- 

^ifo  Q°  ^Qs^&r      cumstances  surrounded  by  a  transparent 

Ji  j  capsule.      The    gelatinous  envelope  —  so- 

Fio.88.-Baciiius  of  Friedlander;     called  capsule — is  not  seen  in  preparations 

o,  from  a  culture;  6,  from  blood  of    made  from  cultures  in  artificial  media,  but 

mouse,  showing  capsule.  (Flugge.)      .  .  .        .. 

is  very  prominent  in  properly  stained  prepa- 
rations from  the  blood  of  an  inoculated  animal.  It  often  has  a  diame- 
ter equal  to  or  greater  than  that  of  the  enclosed  cell,  and  appears  to 
consist  of  a  substance  resembling  mucin,  which  is  soluble  in  water  or 
dilute  alcohol.  Where  several  cells  are  united  in  a  chain  they  may 
all  be  enclosed  in  a  common  envelope,  or  each  may  have  its  own  cap- 
sule. This  capsule  is  not  peculiar  to  Friedlander's  bacillus,  as  he 
at  first  supposed,  but  is  found  in  other  bacilli  and  also  in  the  writer's 
Micrococcus  Pasteuri. 

Friedlander's  bacillus  stains  readily  with  the  aniline  colors,  but 
is  decolorized  by  the  iodine  solution  used  in  Gram's  method.  In 
preparations  from  the  blood  of  an  inoculated  animal,  stained  by  an 
aniline  color,  the  capsule  appears  as  an  unstained  envelope  surround- 
ing the  stained  cell,  but  by  special  treatment  the  capsule  may  also  be 
stained.  Friedlander's  method  is  as  follows  :  The  section  or  cover- 
glass  preparation  is  placed  for  twenty-four  hours  in  a  solution  of 
gentian  violet  and  acetic  acid,  containing  fifty  parts  of  a  concentrated 
alcoholic  solution  of  gentian  violet,  one  hundred  parts  of  distilled 
water,  and  ten  parts  of  acetic  acid.  The  stained  preparation  is 
washed  for  a  minute  or  two  in  a  one-per-cent  solution  of  acetic  acid, 
dehydrated  with  alcohol,  cleared  up  with  oil  of  cloves  or  cedar,  and 
mount.Ml  in  balsam.  The  bacillus  is  quickly  stained  in  dried  cover- 
glass  preparations  by  immersion  in  aniline- water-gentian-violet  solu- 
tion (two  or  three  minutes).  The  stained  preparation  should  be  de- 
colorized by  placing  it  in  absolute  alcohol  for  half  a  minute,  and  then 
washed  in  distilled  water. 


BACTERIA   IN   CROUPOUS   PNEUMONIA. 


309 


Biological  Characters.  —  This  bacillus  does  not,  so  far  as  is 
known,  form  reproductive  spores  ;  it  is  non-motile  and  does  not 
liquefy  gelatin.  It  is  aerobic  and  a  facultative  anaerobic.  In 
gelatin  stick  cultures  it  presents  the  "nail-shaped"  growth  first 
described  by  Friedlander,  which  is  not,  however,  peculiar  to  this 
bacillus.  The  head  of  the  nail  is  formed  by  the 
development  around  the  point  of  entrance  of  the 
inoculating  needle  of  a  rounded,  white  mass  hav- 
ing a  smooth,  shining  surface,  and  its  stem  by  the 
growth  along  the  line  of  puncture.  This  consists 
of  closely  crowded,  opaque,  white,  spherical  colo- 
nies. Gas  bubbles  sometimes  develop  in  gelatin 
cultures,  and  in  old  cultures  the  gelatin  about  the 
line  of  growth  acquires  a  yellowish-brown  color. 
The  growth  in  nutrient  agar  resembles  that  in 
gelatin.  Upon  the  surface  of  blood  serum  abun- 
dant grayish-  white,  viscid  masses  are  developed. 
Upon  potato  the  growth  is  abundant,  quickly  cov- 
ering the  entire  surface  with  a  thick,  yellowish- 
white,  glistening  layer  which  often  contains  gas 
bubbles  when  the  temperature  is  favorable.  Col- 
onies in  gelatin  plates  appear  at  the  end  of  twenty- 
four  hours  as  small,  white  spheres,  which  increase 
rapidly  in  size,  and  upon  the  surface  form  round- 
ed, smooth,  glistening,  white  masses  of  consider- 
able size.  Under  the  microscope  the  colonies  pre- 
sent a  somewhat  irregular  outline  and  a  slightly 

&        J   bacillus;  stick  culture  in 

granular  appearance.    Growth  occurs  at  compara-  gelatin;  end  of  four  days 


tively  low  temperatures—  16°  to  20°  C.—  but  is  more  *etn16°-18°  c' 

rapid  in  the  incubating  oven.    The  thermal  death- 

point,  as  determined  by  the  writer,  is  about  56°  C.     In  the  ordinary 

culture  media  it  retains  its  vitality  for  a  long  time,  and  may  grow 

when  transplanted  to  fresh  culture  material  after  having  been  pre- 

served for  a  year  or  more.     At  a  temperature  of  40°  C.  development 

ceases. 

Pathogenesis.  —  In  Friedlander's  experiments  the  bacillus  from 
pure  cultures,  suspended  in  water,  was  injected  through  the  thoracic 
wall  4nto  the  right  lung  of  dogs,  rabbits,  guinea-pigs,  and  mice. 
Rabbits  proved  to  be  immune  ;  one  dog  out  of  five,  six  guinea-pigs 
out  of  eleven,  and  all  of  the  mice  (thirty-two)  succumbed  to  the 
inoculation.  At  the  autopsy  the  pleural  cavities  were  found  to  con- 
tain a  sero-purulent  fluid  ;  the  lungs  were  intensely  congested,  con- 
tained but  little  air,  and  in  places  showed  limited  areas  of  red  infil- 
tration ;  the  spleen  was  considerably  enlarged  ;  the  bacillus  was 


310  BACTERIA  IN  CROUPOUS   PNEUMONIA. 

found  in  great  numbers  in  the  lungs,  the  fluid  in  the  pleural  cavi- 
ties, and  in  the  blood  obtained  from  the  general  circulation  or  from 
the  various  organs  of  the  body.  Similar  appearances  presented  them- 
selves in  the  case  of  the  guinea-pigs  which  succumbed  to  the  inocu- 
lation. 

These  results  show  that  the  bacillus  under  consideration  is  path- 
ogenic for  mice  and  for  guinea-pigs,  but  they  are  by  no  means 
sufficient  to  prove  that  it  is  capable  of  producing  a  genuine  croupous 
pneumonia  in  man,  and  it  is  still  uncertain  whether  its  occasional 
presence  in  the  exudate  into  the  pulmonary  alveoli  in  cases  of  this 
disease  has  any  etiological  importance. 

8.    MICROCOCCUS  PNEUMONIA   CROUPOS^E. 

Synonyms. — Micrococcus  Pasteuri  (Sternberg)  ;  Micrococcus  of 
sputum  septicaemia  (Frankel)  ;  Diplococcus  pneumonia  (Weichsel- 
baum) ;  Bacillus  septicus  sputigenus  (Fliigge)  ;  Bacillus  salivarius 
septicus  (Biondi)  ;  Lancet-shaped  micrococcus  (Talamon)  ;  Strepto- 
coccus lanceolatus  Pasteuri  (Gameleia). 

Discovered  by  the  present  writer  in  the  blood  of  rabbits  inocu- 
lated subcutaneously  with  his  own  saliva  in  September,  1880  ;  by 
Pasteur  in  the  blood  of  rabbits  inoculated  with  the  saliva  of  a  child 
which  died  of  hydrophobia  in  one  of  the  hospitals  of  Paris  in  De- 
cember, 1880  ;  identified  with  the  micrococcus  in  the  rusty  sputum  of 
pneumonia,  by  comparative  inoculation  and  culture  experiments,  by 
the  writer  in  1885  (paper  published  in  the  American  Journal  of  the 
Medical  Sciences,  July  1st,  1885).  Proved  to  be  the  cause  of  croup- 
ous pneumonia  in  man  by  the  researches  of  Talamon,  Salvioli,  Stern- 
berg,  Frankel,  Weichselbaum,  Netter,  Gameleia,  and  others. 

The  Presence  of  Micrococcus  Pasteuri  in  the  Salivary  Secre- 
tions of  Healthy  Individuals. — In  September,  1880,  while  engaged 
in  investigations  relating  to  the  etiology  of  the  malarial  fevers,  I  in- 
jected a  little  of  my  own  saliva  beneath  the  skin  of  two  rabbits  as  a 
control  experiment.  To  my  surprise  the  animals  died,  and  I  found 
in  their  blood  a  multitude  of  oval  microorganisms,  united  for  the 
most  part  in  pairs,  or  in  chains  of  three  or  four  elements.  These 
experiments  are  recorded  in  my  paper  entitled  "  Experimental  Inves- 
tigations Relating  to  the  Etiology  of  the  Malarial  Fevers/'  published 
in  the  Report  of  the  National  Board  of  Health  for  1881,  pp.  74,  75. 

Following  up  my  experiments  made  in  New  Orleans  (in  Septem- 
ber, 1880),  in  Philadelphia  (January,  1881),  and  in  Baltimore  (March, 
1881),  I  obtained  the  following  results  : 

"  The  saliva  of  four  students,  residents  of  Baltimore  (in  March), 
gave  negative  results  ;  eleven  rabbits  injected  with  the  saliva  of  six 
individuals  in  Philadelphia  (in  January)  gave  eight  deaths  and  three 


BACTERIA  IN  CROUPOUS  PNEUMONIA.  311 

negative  results;  but  in  the  fatal  cases  a  less  degree  of  virulence  was 
shown  in  six  by  a  more  prolonged  period  between  the  date  of  injec- 
tion and  the  date  of  death.  This  was  three  days  in  one,  four  days 
in  four,  and  seven  days  in  one." 

In  a  paper  published  in  the  Journal  of  the  Royal  Microscopical 
Society  (June,  1886)  I  say  : 

"  My  own  earlier  experiments  showed  that  there  is  a  difference  in 
the  pathogenic  potency  of  the  saliva  of  different  individuals,  and  I 
have  since  learned  that  the  saliva  of  the  same  individual  may  differ 
in  this  respect  at  different  times.  Thus  during  the  past  three  years 
injections  of  my  own  saliva  have  not  infrequently  failed  to  cause  a 
fatal  result,  and  in  fatal  cases  death  is  apt  to  occur  after  a  some- 
what longer  interval,  seventy-two  hours  or  more ;  whereas  in  my 
earlier  experiments  the  animals  infallibly  died  within  forty-eight 
hours." 

The  presence  of  my  Micrococcus  Pasteuri  was  demonstrated  in 
the  blood  of  the  rabbits  which  succumbed  to  the  inoculations. 

Claxton,  in  a  series  of  experiments  made  in  Philadelphia  in  1882, 
injected  the  saliva  of  seven  individuals  into  eighteen  rabbits.  Five 
of  these  died  within  five  days,  and  nine  at  a  later  period. 

Frankel,  whose  first  publication  was  made  in  1885,  discovered 
the  presence  of  this  micrococcus  in  his  own  salivary  secretions  in  1883, 
and  has  since  made  extended  and  important  researches  with  refe- 
rence to  it.  The  saliva  of  five  healthy  individuals  and  the  sputa 
of  patients  suffering  from  other  diseases  than  pneumonia,  injected 
into  eighteen  rabbits,  induced  fatal  "  sputum  septicaemia  "  in  three 
only.  When  he  commenced  his  experiments  his  saliva  was  uni- 
formly fatal  to  rabbits,  but  a  year  later  it  was  without  effect. 

Wolf  injected  the  saliva  of  twelve  healthy  individuals,  and  of 
three  patients  with  catarrhal  bronchitis,  into  rabbits,  and  induced 
"  sputum  septicaemia  "  in  three. 

Netter  examined  the  saliva  of  one  hundred  and  sixty-five  healthy 
persons,  by  inoculation  experiments  in  rabbits,  and  demonstrated 
the  presence  of  this  micrococcus  in  fifteen  per  cent  of  the  number. 

Vignal,  in  his  recent  elaborate  paper  upon  the  microorganisms 
of  the  mouth,  says  : 

"  Last  year  I  encountered  this  microbe  continually  in  my  mouth 
during  a  period  of  two  months,  then  it  disappeared,  and  I  did  not 
find  it  again  until  April  of  this  year,  and  then  only  for  fifteen  days, 
when  it  again  disappeared  without  appreciable  cause. " 

The  Presence  of  Micrococcus  Pneumonice  Crouposce  in  Pneu- 
monic Sputum. — Talamon,  in  1883,  demonstrated  the  presence  of  this 
micrococcus  in  pneumonic  sputum,  described  its  morphological  char- 
acters, and  produced  typical  croupous  pneumonia  in  rabbits  by  in- 


312  BACTERIA  IN   CROUPOUS   PNEUMONIA. 

jecting  material  containing  it  into  the  lungs  through  the  thoracic 
walls. 

Salvioli,  in  1884,  demonstrated  its  presence  in  pneumonic  sputum 
by  injections  into  rabbits. 

In  1885  the  writer  made  a  similar  demonstration,  and  by  compara- 
tive experiments  showed  that  the  micrococcus  present  in  the  blood 
of  rabbits  inoculated  with  the  rusty  sputum  of  pneumonia  was  iden- 
tical with  that  which  he  had  discovered  in  1880  in  rabbits  inoculated 
with  his  own  saliva. 

The  same  year  (1885)  A.  Frankel  made  a  similar  demonstration, 
and  published  a  paper  containing  valuable  additions  to  our  knowl- 
edge relating  to  the  biological  characters  of  this  microorganism  (first 
publication  appeared  July  13th,  1885). 

In  1886  Weichselbaum  published  the  results  gf  his  extended  re- 
searches relating  to  the  presence  of  this  micrococcus  in  the  fibrinous 
exudate  of  croupous  pneumonia.  He  obtained  it  in  ninety-four  cases 
(fifty-four  times  in  cultures)  out  of  one  hundred  and  twenty-nine  cases 
examined. 

Wolf  (1887)  found  it  in  sixty-six  cases  out  of  seventy  examined. 

Netter  (1887)  in  seventy-five  per  cent  of  his  cases,  and  in  the  sputum 
of  convalescents  from  pneumonia  in  sixty  per  cent  of  the  cases  ex- 
amined, by  inoculations  into  rabbits. 

Gameleia  (1887)  in  twelve  fatal  cases  of  pneumonia  in  which  he 
collected  material  from  the  lungs  at  the  post-mortem  examination. 

Goldenberg,  whose  researches  were  made  in  Gameleia's  labora- 
tory, found  it  in  pneumonic  sputum  in  forty  consecutive  cases,  by 
inoculations  into  rabbits  and  mice. 

The  Presence  of  Micrococcus  Pneumonice  Crouposce  in  Menin- 
gitis.— Numerous  bacteriologists  have  reported  finding  diplococci  in 
the  pus  of  meningitis,  and  frequently  the  microorganisms  have  been 
fully  identified  as  "  diplococcus  pneumoniae."  Thus  Netter  (1889),  in 
a  resume  of  the  results  of  researches  made  by  him  in  twenty-five 
cases  of  purulent  meningitis,  reports  as  follows  : 

Thirteen  cases  were  examined  microscopically,  by  cultures,  and 
by  inoculations  into  susceptible  animals  ;  six  cases  by  microscopical 
examination  and  experiments  on  animals;  and  the  remainder  only  by 
microscopical  examination.  Four  of  the  cases  were  complicated 
with  purulent  otitis,  six  with  pneumonia,  three  with  ulcerative  endo- 
carditis. The  "  pneumococcus"  was  found  in  sixteen  of  the  twenty- 
five  cases ;  in  four  Streptococcus  pyogenes  was  present ;  in  two 
Diplococcus  intracellularis  meningitidis  of  Weichselbaum  ;  in  one 
Friedlander's  bacillus  ;  in  one  Newmann  and  Schaffer's  motile  ba- 
cillus ;  in  one  a  small  curved  bacillus. 

In  forty-five  cases  collected  from  the  literature  of  the  subject  by 


BACTERIA   IN   CROUPOUS   PNEUMONIA.  313 

Netter  this  micrococcus  was  present  in  twenty-seven,  Streptococcus 
pyogenes  in  six,  and  the  Diplococcus  intracellularis  meningitidis  of 
Weichselbaum  in  ten. 

Monti  (1889),  in  four  cases  of  cerebro-spinal  meningitis,  demon- 
strated the  presence  of  the  same  micrococcus.  In  three  of  his  cases 
pneumonia  was  also  present.  In  two  Staphylococcus  pyogenes  aureus 
was  associated  with  the  "  diplococcus  pneumonias." 

Micrococcus  Pneumonice  Crouposce  in  Ulcerative  Endocar- 
ditis.— Weichselbaum,  in  a  series  of  twenty-nine  cases  examined 
(1888),  found  "  diplococcus  pneumonise"  in  seven. 

Micrococcus  Pneumonice  Crouposce  in  Acute  Abscesses. — In  a 
case  of  parotitis  occurring  as  a  complication  of  croupous  pneumonia 
this  micrococcus  was  obtained  from  the  pus  in  pure  cultures  by  Testi 
(1889);  and  in  another  case  in  which,  as  a  complication  of  pneumonia, 
there  developed  a  purulent  pleuritis,  abscess  of  the  parotid  on  both 
sides,  and  multiple  subcutaneous  abscesses,  the  pus  from  all  of  the 
sources  named  contained  the  "diplococcus"  in  great  numbers,  as 


FIG.  90.  FIG.  91.  FIG.  98. 

FIG.  90.— Micrococcus  pneumonias  crouposse  from  blood  of  rabbit  inoculated  with  normal  human 
saliva  (Dr.  S.).  X  1,000. 

FIG.  91. — Micrococcus  pneumonias  crouposse  from  blood  of  rabbit  inoculated  subcutaneously 
with  fresh  pneumonic  sputum  from  a  patient  in  the  seventh  day  of  the  disease.  X  1,000. 

FIG.  92.— Surface  culture  of  Micrococcus  pneumoniae  crouposse,  on  nutrient  agar,  showing  the 
development  of  long  chains.  X  1,000. 1 

shown  not  only  by  microscopical  examination  but  by  inoculation  into 
rabbits. 

In  a  case  of  tonsillitis  resulting  in  the  formation  of  an  abscess 
Gabbi  (1889)  obtained  the  same  coccus  in  pure  cultures. 

In  otitis  media  this  micrococcus  has  been  found  in  a  consider- 
able number  of  cases  in  the  pus  obtained  by  paracentesis  of  the 
tympanic  membrane,  and  quite  frequently  in  pure  cultures — by  Zau- 
fal  (1889)  in  six  cases;  Levy  and  Schrader  (1889)  in  three  out  of  ten 
cases  in  which  paracentesis  was  performed;  by  Netter  (1889)  in  five 
out  of  eighteen  cases  occurring  in  children. 

Monti  (1889)  and  Belfanti  (1889)  report  cases  of  arthritis  of  the 
wrist  joint,  occurring  as  a  complication  of  pneumonia,  in  which  this 
micrococcus  was  obtained  in  pure  cultures.  Ortmann  and  Samter 

1  The  above  figures  are  from  Dr.  Sternberg's  paper  published  in  the  American 
Journal  of  the  Medical  Sciences  for  July  and  October,  1885. 


314  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

(1889),  in  a  case  of  purulent  inflammation  of  the  shoulder  joint  fol- 
lowing pneumonia  and  pleurisy,  obtained  the  "diplococcus  pneu- 
monise  "  in  pure  cultures. 

Morphology. — Spherical  or  oval  cocci,  usually  united  in  pairs,  or 
in  chains  consisting  of  three  or  four  elements.  Longer  chains,  con- 
taining ten  or  more  elements,  are  frequently  formed,  especially  in 
cultures  upon  the  surface  of  nutrient  agar,  and  in  liquid  media;  it 
may  therefore  be  regarded  as  a  streptococcus.  As  observed  in  the 
blood  of  inoculated  animals  it  is  usually  in  pairs  consisting  of  oval 
or  lance-oval  elements,  which  are  surrounded  by  a  transparent  cap- 
sule. Owing  to  the  elongated  form  of  the  cocci  when  in  active 
growth,  it  has  been  regarded  by  some  authors  as  a  bacillus;  but  in 
cultures  in  liquid  media,  when  development  by  binary  division  has 
ceased,  the  cells  are  spherical,  or  nearly  so,  and  in  cultures  on  the 
surface  of  nutrient  agar  the  individual  cells  more  nearly  approach  a 
spherical  form  than  in  the  blood  of  an  inoculated  animal.  The  "  lan- 
ceolate "  form  was  first  referred  to  by  Talamon,  who  described  it  as 
having  the  form  of  a  grain  of  wheat,  or  even  still  more  elongated 
like  a  grain  of  barley,  as  seen  in  the  fibrinous  exudate  of  croupous 
pneumonia.  *  The  transparent  material  surrounding  the  cells — so- 
called  capsule — is  best  seen  in  stained  preparations  from  the  fibrinous 
exudate  of  croupous  pneumonia  or  from  the  blood  of  an  inoculated 
animal.  It  appears  as  an  unstained  marginal  band  surrounding  the 
elliptical  cells,  and  varies  greatly  as  to  its  extent  in  different  prepara- 
tions. This  capsule  probably  consists  of  a 
substance  resembling  mucin,  and,  being  solu- 
ble in  water,  its  extent  depends  partly  upon 
the  methods  employed  in  preparing  speci- 
mens for  microscopical  examination.  It  is 
occasionally  seen  in  stained  preparations  from 
the  surface  of  cultures  on  blood  serum ;  and 
in  drop  cultures  examined  under  the  micro- 

^P6'   by  usi"g  a  sma11  di^^agm  it  may 
suie,  attached  to  pus  cells  from    be  seen  to  surround  the  cocci  as  a  scarcely 

exudate  in  pleura!  cavity  of     vidihln  Vialn 
inoculated  rabbit.    (Salvioli.)      V1S1^e.  nal°' 

This  micrococcus  stains  readily  with  the 

aniline  colors;  and  also  by  Gram's  method,  which  constitutes  an 
important  character  for  distinguishing  it  from  Friedlander's  ba- 
cillus. 

Biological  Characters. — Grows  in  the  presence  of  oxygen — 
aerobic — but  is  also  a  facultative  anaerobic.  Like  other  micro- 
cocci,  it  has  no  spontaneous  movements.  It  grows  in  a  variety  of 
culture  media  when  they  have  a  slightly  alkaline  reaction,  but  will 
not  develop  in  a  medium  which  contains  the  slightest  trace  of  free 

. 


BACTERIA   IN  CROUPOUS   PNEUMONIA.  315 

acid.  Nor  will  it  grow  at  the  ordinary  room  temperature.  Scanty 
development  may  occur  at  a  temperature  of  22°  to  24°  C.,  but  a 
temperature  of  35°  to  37°  C.  is  most  favorable  for  its  growth,  which 
is  very  rapid  in  a  suitable  liquid  medium.  In  an  infusion  made  from 
the  flesh  of  a  chicken  or  a  rabbit  it  multiplies,  in  the  incubating 
oven,  with  remarkable  rapidity  ;  at  the  end  of  six  to  twelve  hours 
after  inoculation  the  previously  transparent  fluid  will  be  found  to 
present  a  slight  cloudiness  and  to  be  filled  throughout  with  the  cocci 
in  pairs  and  short  chains.  It  does  not  produce  a  milky  opacity  in 
liquid  media,  like  the  pus  cocci,  for  example,  but  the  fluid  becomes 
slightly  clouded  ;  multiplication  ceases  at  the  end  of  about  forty- 
eight  hours  or  less,  and  the  liquid  medium  again  becomes  transpa- 
rent as  a  result  of  the  subsidence  of  the  cocci  to  the  bottom  of  the 
receptacle. 

It  may  be  cultivated  in  flesh-peptone-gelatin,  containing  fifteen 
per  cent  of  gelatin,  at  a  temperature  of  24°  C. ,  or  in  liquefied  gela- 
tin (ten  per  cent)  in  the  incubating  oven. 
In  gelatin  (fifteen  per  cent)  stick  cultures 
small  white  colonies  develop  all  along  the 
line  of  puncture,  and  in  gelatin  plates 
small,  spherical,  slightly  granular,  whitish 
colonies  are  formed  :  the  gelatin  is  not 
liquefied.  In  agar  plates  extremely  mi- 
nute colonies  are  developed  in  the  course 
of  forty-eight  hours,  which  resemble  little, 
transparent  drops  of  fluid,  and  under  the 
microscope  some  of  these  are  observed  to  FIG. 
have  a  compact,  finely  granular  central  coccus  pneumonise  crouposse  upon 
portion  surrounded  by  a  paler,  transparent, 
finely  granular  marginal  zone.  Upon  the 
surface  of  nutrient  agar  or  coagulated  blood  serum  development 
occurs  in  the  form  of  minute,  transparent,  jelly-like  drops,  which 
form  a  thin  layer  along  the  line  of  inoculation  in  "streak  cultures"  ; 
and  in  agar  stick  cultures  the  growth  along  the  line  of  puncture  is 
rather  scanty,  almost  homogeneous,  and  semi-transparent.  Upon 
potato  no  development  occurs,  even  in  the  incubating  oven.  Milk  is 
a  favorable  culture  medium,  and  the  casein  is  coagulated  as  a  result 
of  its  presence. 

It  ceases  to  grow  on  solid  media  at  about  40°  C.,  and  in  favorable 
liquid  media  at  42°  C.  Its  thermal  death-point,  as  determined  by 
the  writer,  is  52°  C.,  the  time  of  exposure  being  ten  minutes.  It 
loses  its  vitality  in  cultures  in  a  comparatively  short  time — four  or 
five  days  on  agar — and  is  very  sensitive  to  the  action  of  germicidal 
agents.  Its  pathogenic  power  also  undergoes  attenuation  very 


316  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

quickly  when  it  is  cultivated  in  artificial  media,  but  may  be  restored 
by  passing  it  through  the  bodies  of  susceptible  animals.  Attenua- 
tion of  virulence  may  also  be  effected  by  exposing  bouillon  cultures 
to  a  temperature  of  42°  C.  for  twenty-four  hours,  or  by  five  days' 
exposure  to  a  temperature  of  41°  C. 

Emmerich  reported  in  1891  to  the  Congress  of  Hygiene  and 
Demography  in  London  the  results  of  experiments  made  by  him 
relating  to  immunity  in  rabbits  and  mice.  Rabbits  were  rendered 
immune  by  the  intravenous  injection  of  a  very  much  diluted  but 
virulent  culture  of  the  micrococcus.  The  flesh  of  these  immune 
rabbits  was  rubbed  up  into  a  fine  paste,  and  the  juices  obtained  by 
compressing  it  in  a  clean,  sterilized  cloth.  This  bloody  juice  was  kept 
for  twelve  hours  at  a  temperature  of  10°  C.,  and  then  sterilized  by 
passing  it  through  a  Pasteur  filter.  Some  of  this  juice  was  injected 
into  a  rabbit,  which  with  twenty-five  others  was  then  made  to  re- 
spire an  atmosphere  charged  with  a  spray  of  a  bouillon  culture  of 
the  micrococcus.  As  a  result  of  this  all  of  the  rabbits  died  except  the 
one  which  had  previously  been  injected  with  the  immunizing  juice. 
In  a  similar  experiment  upon  mice  six  of  these  animals,  which  had 
previously  been  injected  with  the  immunizing  juice,  survived  the  in- 
jection of  a  full  dose  of  a  virulent  culture,  while  a  control  mouse, 
not  previously  injected  with  the  juice,  promptly  died  after  receiving 
the  same  quantity  of  the  virulent  culture. 

The  writer  in  1881,  in  experiments  made  to  determine  the  value 
of  various  disinfectants,  as  tested  upon  this  micrococcus,  obtained 
experimental  evidence  that  its  virulence  is  attenuated  by  the  action 
of  certain  antiseptic  agents.  Commenting  upon  the  results  of  these 
experiments  in  my  chapter  on  "  Attenuation  of  Virus/'  in  "  Bacte- 
ria "(1884),  I  say: 

"Sodium  hyposulphite  and  alcohol  were  the  chemical  reagents  which 
produced  the  result  noted  in  these  experiments  ;  but  it  seems  probable  that 
a  variety  of  antiseptic  substances  will  be  found  to  be  equally  effective  when 
used  in  the  proper  proportion.  Subsequent  experiments  have  shown  that 
neither  of  these  agents  is  capable  of  destroying  the  vitality  of  this  septic 
micrococcus  in  the  proportion  used  (one  per  cent  of  sodium  hyposulphite  or 
one  part  of  ninety-ftve-per-cent  alcohol  to  three  parts  of  virus),  and  that 
both  have  a  restraining  influence  upon  the  development  of  this  microorgan- 
ism in  culture  fluids." 

The  following  results  were  obtained  by  the  writer  in  his  experi- 
ments (1881  and  1883)  to  determine  the  germicidal  and  antiseptic 
value  of  the  agents  named,  as  tested  upon  this  micrococcus. 

Alcohol. — A  twenty-four-per-cent  solution  was  effective  upon 
bouillon  cultures  in  two  hours. 

Boric  Acid. — A  saturated  solution  failed  to  destroy  vitality  after 
two  hours'  exposure,  but  1  :  400  restrained  development. 


BACTERIA   IN  CROUPOUS  PNEUMONIA.  317 

Carbolic  Acid. — A  one-per-cent  solution  destroys  vitality  in  two 
hours,  and  1  :  500  restrains  development. 

Cupric  Sulphate  destroys  the  virulence  of  the  coccus  in  the 
blood  of  a  rabbit  in  the  proportion  of  1  : 400  in  half  an  hour. 

Ferric  Sulphate  failed  to  destroy  vitality  in  a  saturated  solution, 
but  restrained  development  in  the  proportion  of  1  :  200. 

Hydrochloric  Acid  destroys  the  virulence  of  the  blood  of  a  rab- 
bit containing  this  micrococcus  in  the  proportion  of  1  :  200. 

Iodine,  in  aqueous  solution  with  potassium  iodide,  destroys  vital- 
ity in  the  proportion  of  1: 1,000  and  prevents  development  in  1:  4,000. 

Mercuric  Chloride. — One  part  in  forty  thousand  prevents  the 
development  of  this  micrococcus,  and  1  :  20,000  was  found  to  destroy 
vitality  in  two  hours. 

Nitric  Acid. — One  part  in  four  hundred  destroyed  the  virulence 
of  rabbit's  blood  containing  this  micrococcus. 

Caustic  Potash. — A  two-per-cent  solution  destroyed  vitality  in 
two  hours. 

Potassium  Permanganate. — A  two-per-cent  solution  destroyed 
the  virulence  of  rabbit's  blood  containing  this  coccus. 

Salicylic  Acid,  dissolved  by  the  addition  of  sodium  biborate. — 
A  solution  of  1  :  400  prevented  development. 

Sulphuric  Acid. — One  part  in  two  hundred  destroys  vitality,  and 
1  :  800  prevents  development. 

In  a  paper  by  Bordoni-Uffreduzzi  relating  to  the  resisting  power 
of  pneumonic  virus  for  desiccation  and  light,  the  following  results  are 
given :  Pneumonic  sputum  attached  to  cloths,  when  dried  in  the  air 
and  exposed  to  diffuse  daylight,  retained  its  virulence,  as  shown  by 
injection  in  rabbits,  for  a  period  of  nineteen  days  in  one  series  of  ex- 
periments and  for  fifty-five  days  in  another.  Exposed  to  direct  sun- 
light the  same  material  retained  its  virulence  after  twelve  hours' 
exposure.  Cultures  have  far  less  resistance,  and  the  protection 
afforded  by  the  dried  albuminous  material  in  which  the  micrococci 
were  embedded,  in  the  experiments  referred  to,  probably  accounts 
for  the  virulence  being  retained  so  long  a  time. 

Kruse  and  Pansini  (1892)  have  published  an  elaborate  paper  giv- 
ing an  account  of  their  researches  relating  to  "diplococcus  pneumo- 
niae  "  and  allied  streptococci.  We  give  below  a  summary  statement 
of  their  results : 

Many  varieties  were  obtained  by  the  observers  named  in  their  cultures 
from  various  sources — from  the  lungs  of  individuals  dead  from  pneumonia, 
from  pleuritic  exudate,  from  pneumonic  sputa,  from  bronchitic  sputa,  from 
the  saliva  of  healthy  persons,  from  the  secretion  in  a  case  of  subacute  nasal 
catarrh ,  from  the  urine  of  a  patient  with  nephritis. 

Pure  cultures  were  obtained  by  the  use  of  agar  plates  or  by  inoculations 
into  rabbits.  In  all  about  thirty  varieties  were  obtained  and  cultivated 


318  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

through  many  successive  generations.  As  a  rule,  the  different  varieties, 
which* at  first  were  seen  to  have  the  form  of  diplococci,  when  cultivated  for 
a  length  of  time  in  artificial  media  presented  the  form  of  streptococci  ;  and 
the  elements  which  at  first  were  lancet-shaped  showed  a  tendency  to  become 
spherical. 

The  more  virulent  varieties  usually  presented  the  form  of  diplococci 
with  lancet-shaped  elements,  or  of  short  chains.  A  variety  which  formed 
long  chains  could  be  pronounced,  in  advance  of  the  experiments  on  animals, 
to  possess  comparatively  little  virulence.  When  by  inoculations  in  animals 
the  virulence  of  such  a  variety  was  restored,  the  tendency  to  form  chains 
was  less  pronounced. 

Although,  as  a  rule,  no  development  occurs  at  20°  C.,  certain  varieties 
were  obtained  which,  after  long  cultivation  in  artificial  media,  showed  a  de- 
cided growth  at  18°  C. 

Decided  differences  were  shown  by  the  cultures  from  various  sources  as 
regards  their  growth  in  rnilk.  Out  of  eighty-four  cultures  from  various 
sources  eleven  did  not  produce  coagulation.  As  a  rule,  cultures  which  caused 
coagulation  of  milk  were  virulent  for  rabbits,  and  when  such  cultures  lost 
their  virulence  they  usually  lost  at  the  same  time  the  power  of  coagulating 
milk.  Virulent  cultures  die  out  sooner  than  those  which  have  become  at- 
tenuated by  continuous  cultivation  in  artificial  media;  the  first,  on  the  sur- 
face of  agar,  usually  fail  to  grow  at  the  end  of  a  week,  while  the  attenuated 
cultures  may  survive  for  three  weeks  or  more. 

Pathogenesis. — This  micrococcus  is  very  pathogenic  for  mice  and 
for  rabbits,  less  so  for  guinea-pigs.  The  injection  of  a  minute  quan- 
tity— 0.2  cubic  centimetre  or  less — of  a  virulent  culture  beneath  the 
skin  of  a  rabbit  or  a  mouse  usually  results  in  the  death  of  the  animal 
in  from  twenty-four  to  forty-eight  hours.  The  following  is  from  the 
writer's  first  published  paper  (1881),  and  refers  to  the  pathological 
appearances  in  rabbits : 

"  The  course  of  the  disease  and  the  post-mortem  appearances  indicate  that 
it  is  a  form  of  septicaemia.  Immediately  after  the  injection  there  is  a  rise  of 
temperature,  which  in  a  few  hours  may  reach  2°  to  3°  C.  (3.6°  to  5.4°  F.); 
the  temperature  subsequently  falls,  and  shortly  before  death  is  often  several 
degrees  below  the  normal.  There  is  loss  of  appetite  and  marked  debility 
after  twenty -four  hours,  and  the  animal  commonly  dies  during  the  second 
night  or  early  in  the  morning  of  the  second  day  after  the  injection.  Death 
occurs  still  more  quickly  when  the  blood  from  a  rabbit  recently  dead  is  in- 
jected. Not  infrequently  convulsions  immediately  precede  death. 

4 'The  most  marked  pathological  appearance  is  a  diffuse  inflammatory 
oadema  or  cellulitis,  extending  in  all  directions  from  the  point  of  injection, 
but  especially  to  the  dependent  portions  of  the  body.  Occasionally  there  is 
a  little  pus  near  the  puncture,  but  usually  death  occurs  before  the  cellulitis 
reaches  the  point  of  producing  pus.  The  subcutaneous  connective  tissue 
contains  a  quantity  of  bloody  serum,  which  possesses  virulent  properties  and 
which  contains  a  multitude  of  micrococci.  There  is  usui  lly  more  or  less  in- 
flammatory adhesion  of  the  integument  to  the  subjacent  tissues.  The  liver 
is  sometimes  dark-colored  and  gorged  with  blood,  but  more  frequently  it  is 
of  a  lighter  color  than  normal  and  contains  much  fat.  The  spleen  is  either 
normal  in  appearance  or  enlarged  and  dark-colored.  Changes  in  this  organ 
are  more  marked  in  those  cus.-s  which  are  of  the  longest  duration. 

The  blood  commonly  contains  an  immense  number  of  micrococci,  usually 
•joined  in  pairs  and  having  a  diameter  of  about  0.5  //.  These  are  found  in 
blood  drawn  tmm  superficial  veins,  from  arteries,  and  from  the  cavities  of 
the  heart  immediately  after  death,  and  in  a  few  cases  their  presence  has  been 


BACTERIA   IN   CROUPOUS    PNEUMONIA.  319 

verified  during  life.  Observations  thus  far  made,  however,  indicate  that  it 
is  only  during  the  last  hours  of  life  that  these  parasites  multiply  in  the  cir- 
culating fluid,  and  in  a  certain  proportion  of  the  cases  a  careful  search  has 
failed  to  reveal  their  presence  in  the  hlood  in  post-mortem  examinations 
made  immediately  after  the  death  of  the  animal." 

In  animals  which  are  not  examined  until  some  hours  after  death 
a  considerable  increase  in  the  number  of  micrococci  occurs  post  mor- 
tem. The  fact  that  this  micrococcus  varies  very  much  as  to  its 
pathogenic  power,  as  a  result  of  conditions  relating  to  the  medium  in 
which  it  develops,  was  insisted  upon  in  my  first  published  paper,  and 
has  been  fully  established  by  later  researches  (Frankel,  Gameleia). 
Susceptible  animals  inoculated  with  attenuated  cultures  acquire  an 
immunity  against  virulent  cultures. 


FIG.  9).— Micrococcus  pneumonias  crouposae  in  blood  of  rabbit  inoculated  with  pneumonic  spu- 
tum,    x  1,000. 

In  dogs  subcutaneous  injections  usually  give  a  negative  result, 
or  at  most  a  small  abscess  forms  at  the  point  of  inoculation.  In  a 
single  experiment,  however,  the  writer  has  seen  a  fatal  result  in  a 
dog  from  the  injection  of  one  cubic  centimetre  of  bloody  serum  from 
the  subcutaneous  connective  tissue  of  a  rabbit  recently  dead.  This 
shows  the  intense  virulence  of  the  micrococcus  when  cultivated  in 
the  body  of  this  animal.  Pneumonia  never  results  from  subcutane- 
ous injections  into  susceptible  animals,  but  injections  made  through 
the  thoracic  walls  into  the  substance  of  the  lung  may  induce  a  typi- 
cal fibrinous  pneumonia.  This  was  first  demonstrated  by  Talamon 
(1883),  who  injected  the  fibrinous  exudate  of  croupous  pneumonia, 
obtained  after  death,  or  drawn  during  life  by  means  of  a  Pravaz 
syringe  from  the  hepatized  portions  of  the  lung,  into  the  lungs  of 


320  BACTERIA  IN  CROUPOUS  PNEUMONIA. 

rabbits.  According  to  See,  eight  out  of  twenty  animals  experi- 
mented upon  exhibited  "a  veritable  lobar,  fibrinous  pneumonia, 
with  pleurisy  and  pericarditis  of  the  same  nature."  Gameleia  has 
also  induced  pneumonia  in  a  large  number  of  rabbits,  and  also  in  the 
dog  and  the  sheep,  by  injections  directly  into  the  pulmonary  tissue. 
Sheep  were  found  to  survive  subcutaneous  inoculations,  unless  very 
large  doses  (five  cubic  centimetres)  of  the  most  potent  virus  were  in- 
jected. But  intrapuhnonary  inoculations  invariably  induced  a  typi- 
cal fibrinous  pneumonia  which  usually  proved  fatal.  In  dogs  simi- 
lar injections  gave  rise  to  a  "frank,  fibrinous  pneumonia  which 
rarely  proved  fatal,  recovery  usually  occurring  in  from  ten  to  fifteen 
days,  after  the  animal  had  passed  through  the  stages  of  red  and 
gray  hepatization  characteristic  of  this  affection  in  man." 

Monti  claims  to  have  produced  typical  pneumonia  in  rabbits  by 
injecting  cultures  of  this  micrococcus  into  the  trachea. 

From  the  evidence  obtained  in  these  experimental  inoculations, 
and  that  recorded  relating  to  the  presence  of  this  micrococcus  in  the 
fibrinous  exudate  of  croupous  pneumonia,  we  are  justified  in  con- 
cluding that  it  is  the  usual  cause  of  this  disease,  and  consequently 
have  described  it  under  the  name  Micrococcous  pneumonise  crou- 
posae.  We  prefer  this  to  the  name  commonly  employed  by  German 
authors — "  diplococcus  pneumonise" — because  this  micrococcus,  al- 
though commonly  seen  in  pairs,  forms  numerous  short  chains  of 
three  or  four  elements  in  cultures  in  liquid  media,  and  upon  the  sur- 
face of  nutrient  agar  may  grow  out  into  long  chains.  It  would, 
therefore,  more  properly  be  called  a  streptococcus  than  a  diplococcus. 

G.  and  F.  Klemperer,  in  1891,  published  an  important  memoir 
relating  to  the  pathogenic  action  of  this  micrococcus.  They  suc- 
ceeded in  conferring  immunity  upon  susceptible  animals  by  inocu- 
lating them  with  filtered  cultures  of  the  micrococcus,  and  in  some 
instances  this  immunity  had  a  duration  of  six  months.  A  curi- 
ous fact  developed  in  their  researches  was  that  the  potency  of 
the  substance  contained  in  the  filtered  cultures  was  increased  by 
subjecting  these  to  a  temperature  of  41°  to  42°  C.  for  three  or  four 
days,  or  to  a  higher  temperature  (60°  C.)  for  an  hour  or  two.  When 
injected  into  a  vein  after  being  subjected  to  such  a  temperature  im- 
111  unity  was  complete  at  the  end  of  three  or  four  days  ;  but  the  same 
material  not  so  heated  required  larger  doses  and  a  considerably 
longer  time  (fourteen  days)  to  confer  immunity  upon  a  susceptible 
animal.  Tlir  nmvarinrd  material  caused  a  considerable  elevation  of 
temperature,  lasting  for  some  days.  The  authors  mentioned  con- 
clude from  their  investigations  that  the  toxic  substance  present  in 
cultures  of  Micrococcus  pneumonise  crouposse  is  a  proteid  substance, 
which  they  propose  to  call  pneumotoxin.  The  substance  produced 


BACTERIA   IN   CROUPOUS   PNEUMONIA.  321 

in  the  body  of  an  immune  animal,  as  a  result  of  protective  inocula- 
tions, upon  which  the  immunity  of  these  animals  depends,  is  also  a 
proteid,  which  they  call  anti-pneumotoxin.  This  they  isolated  from 
the  blood  serum  of  immune  animals.  By  experiment  they  were  able 
to  demonstrate  that  the  blood  serum  containing  this  protective  pro- 
teid, when  injected  into  other  animals,  rendered  them  immune  ;  and 
also  that  it  arrested  the  progress  of  the  infectious  malady  induced  by 
inoculating  susceptible  animals  with  virulent  cultures  of  the  micro- 
coccus.  When  injected  into  the  circulation  of  an  infected  animal 
its  curative  action  was  manifested  by  a  considerable  reduction  of 
the  body  temperature. 

While  the  micrococcus  of  pneumonia  is  not  usually  seen  in  the 
blood  in  cases  of  pneumonia  it  is  probably  present  in  small  numbers, 
and  secondary  infection  of  the  kidneys  appears  to  be  a  common  occur- 
rence. Thus  Frankel  and  Reiche  (1894)  report  that  in  twenty-two 
cases  out  of  twenty-four  in  which  they  had  an  opportunity  to  exam- 
ine the  kidneys,  this  micrococcus  was  present.  It  was  found  espe- 
cially in  the  larger  branches  of  the  veins  and  arteries,  but  also  in  the 
intertubular  vessels  and  the  glomeruli.  The  kidneys  gave  evidence 
of  degenerative  changes,  and  it  is  probable  that  the  "  pneumococcus  " 
would  have  been  found  in  the  urine  of  some  of  these  cases  if  a  bac- 
teriological examination  had  been  made  during  life. 

21 


VI. 

PATHOGENIC  MICROCOCCI  NOT  DESCRIBED  IN 
SECTIONS  IV.   AND  V. 

9.     DIPLOCOCCUS   INTRACELLULARIS   MENINGITIDIS. 

DISCOVERED  by  Weichselbaum  (1887)  in  the  exudate  of  cerebro 
spinal  meningitis  (six  cases),  for  the  most  part  within  the  cells. 

Morphology. — Micrococci,  usually  united  in  pairs,  in  groups  of 
four,  or  in  little  masses ;  sometimes  solitary  and  larger  (probably 
being  upon  the  point  of  dividing).  Distinguished  by  their  presence 
in  the  interior  of  pus  cells  in  the  exudate,  in  this  respect  resembling 
the  gonococcus. 

Stain  best  with  Loffler's  alkaline  solution  of  methylene  blue. 
Do  not  retain  their  color  when  treated  with  iodine  solution  (Gram's 
method). 

Biological  Characters. — This  micrococcus  does  not  grow  at  the 
room  temperature,  but  upon  nutrient  agar  an  abundant  development 
occurs  in  the  incubating  oven.  Upon  the  surface  of  agar  a  tolerably 
luxuriant,  viscid  growth,  which  by  reflected  light  is  gray  and  by 
transmitted  light  grayish- white  ;  along  the  line  of  puncture  growth 
occurs  only  near  the  surface,  indicating  that  this  micrococcus  will 
not  grow  in  the  absence  of  oxygen.  Upon  plates  made  from  agar- 
agar  (one  per  cent)  and  gelatin  (two  per  cent)  very  small  colonies  are 
formed  in  the  interior  of  the  mass,  and  larger  ones,  of  a  grayish 
color,  on  the  surface.  The  former,  under  the  microscope,  are  seen  to 
be  round  or  slightly  irregular,  finely  granular,  and  of  a  yellowish- 
brown  color.  The  superficial  colonies  have  a  yellowish-brown  nu- 
cleus, surrounded  by  a  more  transparent  zone.  The  growth  upon 
coagulated  blood  serum  is  very  scanty,  as  is  that  in  bouillon ;  no 
growth  occurs  upon  potato.  This  micrococcus  quickly  loses  its  power 
of  reproduction  in  artificial  cultures — within  six  days — and  should 
U>  transj.lanltMl  to  {'roll  material  at  short  intervals—  t\vo  days. 

Pathogenesis. — Mice  are  especially  susceptible,  and  usually  die 
within  forty-eight  hours  after  inoculation.  Also  pathogenic  for 
guinea-pigs,  rabbits,  and  dogs. 


PATHOGENIC    MICROCOCCI   NOT    HERETOFORE   DESCRIBED.      323 

10.    STAPHYLOCOCCUS    SALIVARIUS    PYOGENES. 

Obtained  by  Biondi  (1887)  from  an.  inoculation  abscess  in  a  guinea-pig 
injected  subcutaneously  with,  saliva  from  a  child  suffering  from  scarlatina 
anginosa. 

Morphology. — Spherical  cocci,  0.3  to  0.5.  u  in  diameter,  usually  solitary  in 
the  pus  of  abscesses  or  in  irregular  agglomerations. 

Stains  best  by  Gram's  method. 

Biological  Characters. — Grows  at  a  comparatively  low  temperature 
(12°  to  14°  C.),  and  more  rapidly  in  the  incubating  oven.  In  gelatin  stick 
cultures,  at  the  room  temperature,  growth  occurs  along  the  line  of  punc- 
ture, and  at  the  end  of  eight  days  liquefaction  commences  in  the  form  of 
a  funnel,  at  the  bottom  of  which  little,  white,  shining  masses  accumu- 
late, while  at  the  surface  of  the  liquefied  gelatin  a  white,  viscid  layer  forms. 
In  gelatin  plates  spherical,  well-defined,  opalescent,  whitish  colonies  are 
formed,  which  cause  a  tardy  liquefaction  of  the  surrounding  gelatin.  Upon 
agar-agar  the  growth  is  rapid,  in  the  form  of  a  thick  layer  along  the  line  of 
inoculation  in  streak  cultures,  which  has  a  breadth  of  about  one  millimetre 
at  the  end  of  twenty-four  hours  in  the  incubating  oven,  and  presents  an 
orange-yellow  color  at  the  centre,  fading  out  to  white  at  the  margins.  The 
yellow  color  is  not  by  any  means  as  pronounced  as  in  similar  cultures  of 
Staphylococcus  pyogenes  aureus,  and  liquefaction  of  gelatin  is  much  slower. 

Pathogenesis. — Produces  a  local  abscess  when  inoculated  into  dogs,  rab- 
bits, guinea-pigs,  or  mice.  When  injected  into  the  anterior  chamber  of  the 
eye  of  rabbits,  hypopyon,  followed  by  spontaneous  perforation  of  the  cor- 
nea, resulted.  Injected  into  the  circulation  of  a  guinea-pig  (0.2  to  0.4  cubic 
centimetre)  it  gave  rise  to  general  infection,  and  death  at  the  end  of  eight  to 
ten  days. 

11.    MICROCOCCUS   OF   PROGRESSIVE   TISSUE   NECROSIS   IN   MICE. 

Obtained  by  Koch  (1879)  from  mice  inoculated  subcutaneously  with  putrid 
blood. 

Morphology. — Round  cells,  0.5//  in  diameter,  united  in  chains,  or  at  times 
in  crowded  masses. 

Biological  Characters  not  given. 

Pathogenesis. — Causes  necrosis  of  the  tissues  in  the  vicinity  of  the  point 
of  inoculation  in  mice,  which  extends  rapidly  and  causes  the  death  of  the 
animal  in  about  three  days.  The  blood  and  internal  organs  remain  free  from 
micrococci.  (Possibly  a  very  pathogenic  variety  of  Streptococcus  pyogenes?) 

12.   MICROCOCCUS  OF  PROGRESSIVE  ABSCESS  FORMATION  IN 

RABBITS. 

Obtained  by  Koch  (1879)  from  rabbits 
inoculated  with  putrid  blood. 

Morphology.—  Minute  cocci,  about  0.15  u 
in  diameter,  usually  associated  in  thick, 
cloud-like  zooglcea  masses. 

Biological  Characters  not  given, 

Pathogenesis. — In  rabbits  an  extensive 
abscess  forms  inthe  vicinity  of  the  point  of  in- 
oculation, and  the  animal  dies  in  about  twelve 
days.  The  walls  of  the  abscess  are  formed  of  a 
thin  layer  of  micrococci  associated  in  zoog- 
loea masses;  the  interior  contains  finely  gran- 
ular, cheesy  material,  in  which  the  cocci  ap- 
pear to  have  degenerated  and  perished.  The 
contents  of  the  abscess  injected  into  other  Fro.  96.— Micrococcus  of  progressive 

rahhifq  rirnr1iir>f>  a  «iTYiilaT«  i^snlt       Thp  mWn-      tissue  necrosis  in  mice;  section  of  the 
6  a  SimUar  result,      ine  miCl  cartilage  cells;  5,  streptococci. 

coccus  does  not  invade  the  blood.  CKoch.) 


PATHOGENIC   MICKOCOCCI 


Fio.  97.  —  Micrococcus  of 
pyaemia  in  rabbits,  in  capil- 
lary from  the  cortical  portion 
of  the  kidney.  X700.  (Koch.) 


13.      MICROCOCCUS  OF  PY^MIA  IN  RABBITS. 

Obtained  by  Koch  (1879)  in  rabbits  inoculated 
subcutaneously  with  putrefying  flesh  infusion. 

Morphology. — Round  cells,  0.25  u  in  diameter, 
solitary  or  in  pairs,  which  usually  surround  tHe 
blood  corpuscles  in  a  characteristic  manner. 

Biological  Characters  not  given. 

Pathogenesis. — When  injected  subcutaneously 
in  rabbits  the  blood  is  invaded  and  death  occurs 
from  general  infection.  At  the  autopsv  a  puru- 
lent infiltration  is  found  at  the  point  of  injection, 
there  is  peritonitis,  and  metastatic  abscesses  are 
found  in  the  lungs  and  liver.  Numerous  micro- 
cocci,  closely  surrounding  the  blood  corpuscles, 
are  found  in  the  capillaries  of  the  various  organs, 
the  blood  of  the  heart,  etc.  Two  or  three  drops  of 
blood  from  the  heart  of  a  rabbit  recently  dead,  in- 
jected into  another  animal  of  the  same  species, 
cause  its  death  in  about  forty  hours. 


14.      MICROCOCCUS  OF  SEPTICAEMIA  IN  RABBITS. 

Obtained  by  Koch  (1879)  from  rabbits  inoculated  subcutaneously  with 
putrefying  flesh  infusion. 

Morphology. — Oval  cells,  haying  a  long  diameter  of  0.8  to  1.0  /*. 

Biological  Characters  not  given. 

Pathogenesis. — Produces  general  infection  and  death  in  rabbits  and  mice. 
At  the  autopsy  slight  oedema  is  observed  at  the  point  of  inoculation ;  the 
spleen  is  greatly  enlarged ;  no  peritonitis  and  no  embolic  processes  are  found, 
such  as  characterize  the  pathogenic  action  of  the  last-described  species  (No. 
13) ;  nor  do  the  cocci  accumulate  around  the  red  blood  corpuscles.  They  are 
found  in  the  capillaries  of  the  various  organs  in  masses,  and  especially  in 
the  glomeruli  of  the  kidneys. 

15.      MICROCOCCUS  SALIVARIUS  SEPTICUS. 

Obtained  by  Biondi  (1887)  from  the  saliva  of  a  case  of  puerperal  septicae- 
mia, by  inoculations  into  animals. 

Morphology.— Spherical  or  slightly  oval  cocci,  which,  when  in  rapid  mul- 
tiplication, show  slight  lateral  protrusions. 

Biological  Characters. — Grows  in  nutrient  gelatin  or  agar  at  a  tem- 
perature of  18°  to  20°  C.,  and  more  rapidly  in  the  incubating  oven.  Does  not 
liquefy  gelatin.  In  gelatin  plates  forms  spherical,  grayish- white  colonies, 
which  may  acquire  a  dark  color.  In  gelatin  stick  cultures  grows  along  the 
.line  of  puncture  in  the  form  of  a  column  made  up  of  crowded  white  colo- 
nies. Very  scanty  growth  on  potato. 

Stains  with  all  the  aniline  colors  and  by  Gram's  method. 

Pathogenesis.—  Produces  general  infection  and  death  in  from  four  to  six 
days  when  inoculated  into  mice,  guinea-pigs,  or  rabbits.  The  cocci  are 
found  in  great  numbers,  often  assembled  in  masses,  in  the  capillaries  of  the 
various  organs,  but  no  evidence  of  inflammatory  reaction  of  the  tissues  is  to 
be  observed. 

16.      MICROCOCCUS  SUBFLAVUS   (Flligge). 

Synonym.— Yellowish-white  diplococcus  (Bumm). 

Obtained  by  Bumm  (1885)  from  the  lochial  discharge  of  puerperal  women 
and  from  vaginal  mucus.  Has  also  been  obtained  from  the  urine  in  cases 


NOT   DESCRIBED   IN   SECTIONS  IV.    AND  V.  325 

of  vesical  catarrh,  and  in  the  vesicles  of  pemphigus  ;  also  by  Frankel  in  the 
vaginal  secretion  of  children  suffering-  from  colpitis  not  of  gonorrhceal  origin. 

Morphology.—  Diplococci,  associated  in  biscuit-shaped  pairs,  separated  by 
a  cleft,  and  closely  resembling  the  gonococcus  of  Neisser.  Cells  from  0.5  to 
1.5  n  in  diameter. 

Stains  with  the  aniline  colors  and  by  Gram's  method — by  which  char- 
acter it  may  be  distinguished  from  the  micrococcus  of  gonorrhoea. 

Biological  Characters. — Grows  at  the  room  temperature  upon  the  surface 
of  nutrient  gelatin;  small,  grayish- white  colonies  appear  along  the  line  of 
inoculation  at  the  end  of  twenty-four  hours,  which  later  form  a  confluent 
layer,  first  of  a  pale  yellow  and  finally  of  an  ocherous  color.  In  the  course 
of  a  few  days  liquefaction  of  the  gelatin  commences  in  the  vicinity  of  the 
growth.  Coagulated  blood  serum  is  also  liquefied  by  this  micrococcus. 

Pathogenesis. — Inoculations  upon  mucous  membranes  susceptible  to  gon- 
orrhceal infection  are  without  result.  But  by  injecting  the  diplococcus  from 
pure  cultures,  in  suspension  in  distilled  water,  beneath  the  skin  in  man, 
Bumm  obtained  as  a  result  local  abscess  formation — abscesses  varying  in 
size  from  that  of  a  pigeon's  egg  to  that  of  a  man's  fist.  The  diplococcus  was 
present  in  great  numbers  in  the  pus  of  these  abscesses. 

17.     MICROCOCCUS  OF  TRACHOMA  (?). 

Obtained  by  Sattler  (1885)  from  the  contents  of  the  tuachomatous  follicles 
in  cases  of  Egyptian  ophthalmia;  also  by  Michel  (1886),  who  has  given  a 
more  exact  description  of  this  micrococcus,  and  has  made  inoculation  experi- 
ments which  he  believes  establish  its  etiological  relation  to  the  form  of  oph- 
thalmia with  which  it  is  associated  (?). 

Morphology.  — Very  small,  biscuit-shaped  micro-cocci,  in  pairs — diplococci 
— separated  by  a  very  narrow  dividing  line.  (This  description  would  apply 
to  some  of  the  more  common  pus  cocci,  e.g.,  Stapbylococcus  pyogenes  aureus, 
which  have  also  been  shown  to  consist  of  two  hemispherical  halves  separated 
by  a  narrow  line  of  division.) 

Biological  Characters. — Grows  slowly  upon  nutrient  gelatin  at  the  room 
temperature,  and  does  not  liquefy  this  medium,  upon  the  surface  of  which 
a  grayish-white,  broadly  extended,  glistening  layer  is  formed,  which  later 
has  a  yellowish  tint  and  tulip-shaped  margins.  Spherical  colonies  are  formed 
along  the  line  of  puncture,  which  are  arranged  in  a  linear  series,  like  a 
chaplet.  In  blood  serum  it  grows  along  the  line  of  puncture  as  a  white, 
band-like  stripe,  which  subsequently  spreads  out  in  the  form  of  white  clouds. 
The  growth  is  more  rapid  upon  nutrient  agar  or  blood  serum  in  the  incur 
bating  oven.  The  development  upon  potato  is  very  scanty.  The  cultures 
are  viscid,  drawing  out  into  long  threads  when  touched  with  a  platinum 
needle.  This  micrococcus  does  not  grow  in  the  absence  of  oxygen — aerobic. 

Stains  by  the  aniline  colors  and  by  Gram's  method. 

Pathogenesis. — Not  pathogenic  for  rabbits  when  injected  subcutaneously 
or  into  the  anterior  chamber  of  the  eye ;  but,  according  to  Sattler  and  to 
Michel,  when  inoculated  by  puncture  into  the  conjunctivse  in  man  it  causes 
a  follicular  inflammation  resulting  in  typical  trachoma.  But  Michel  was 
not  able  to  demonstrate  the  presence  of  this  micrococcus  in  all  of  his  cases, 
and  extensive  researches  made  since  by  Baumgarten  and  by  Kartulis  (1887) 
show  that  in  many  cases  of  trachoma,  and  even  in  Egyptian  ophthalmia 
(Kartulis) ,  it  cannot  be  found.  According  to  the  last-named  author,  the  viru- 
lent ophthalmia  which  prevails  in  Egypt  is  gonorrhceal  in  its  origin,  and  he 
has  demonstrated  the  presence  of  the  gonococcus  in  a  large  series  of  cases. 
A  milder,  but  infectious,  acute  catarrhal  conjunctivitis  is  characterized  by 
the  presence  of  a  minute  bacillus,  resembling  the  bacillus  of  mouse  septi- 
caemia, and  found  in  the  pus  cells.  A  third  group  of  chronic  cases  with 
trachoma,  in  the  researches  of  Kartulis,  failed  to  show  the  presence  of  Sat- 
tler's  trachoma  coccus  or  any  other  microorganisms  in  the  contents  of  the 
diseased  follicles. 


326  PATHOGENIC   MICROCOCCI 

18.    MICROCOCCUS  TETRAGENUS. 

First  described  by  Gaffky  (Fliigge).  Obtained  by  Koch  and 
Gaffky  (1881)  from  a  cavity  in  the  lung  in  a  case  of  pulmonary 
phthisis.  Since  found  occasionally  in  normal  saliva  (three  times  in 
fifty  persons  examined  by  Biondi),  and  in  the  pus  of  acute  abscesses 
(Steinhaus,  Park,  Vangel).  Rather  common  in  the  sputum  of  phthi- 
sical cases. 

Morphology. — Micrococci,  having  a  diameter  of  about  one  ^, 
which  divide  in  two  directions,  forming  tetrads,  which  are  enclosed 
in  a  transparent,  jelly-like  envelope— especially  well  developed  as 
seen  in  the  blood  and  tissues  of  inoculated  animals.  In  cultures  the 
cocci  are  seen  in  the  various  stages  of  division,  as  large  single  cells, 


Fio.  98  — Micrococcus  tetragenus;  section  of  lung  of  mouse.    X  800.    (Flugge.) 


pairs  of  oval  elements,  or  groups  of  four  resulting  from  the  trans- 
verse division  of  these  latter. 

Stains  quickly  with  aniline  colors,  and  in  preparations  from  the 
blood  of  an  inoculated  animal  the  transparent  envelope  may  also  be 
feebly  stained.  Stains  also  by  Gram's  method. 

Biological  Characters. — This  micrococcus  grows,  rather  slowly, 
in  nutrient  gelatin  at  the  ordinary  room  temperature,  without  lique- 
faction of  the  gelatin.  Upon  gelatin  plates  small  white  colonies  are 
developed  in  from  twenty-four  to  forty-eight  hours,  which  under  the 
microscope,  with  a  low  power,  are  seen  to  be  spherical  or  lemon- 
shapnK  finely  -rantilar.  and  with  a  inulhcrry-likc  surface.  When 
they  come  to  the  surface  they  form  white,  elevated,  and  rather  thick 
masses  having  a  diameter  of  one  to  two  millimetres.  In  gelatin 
Stick  cultures  a  broad  and  thick  white  mass  forms  upon  the  surface, 


NOT   DESCRIBED   IN  SECTIONS   IV.    AND   V.  327 

and  along  the  line  of  puncture  a  series  of  round,  milk-white  or  yel- 
lowish masses  form,  which  usually  remain  distinct,  but  may  become 
confluent.  Upon  the  surface  of  agar  the  growth  is  similar  to  that 
upon  gelatin,  or  in  streak  inoculations  may  consist  of  a  series  of 
spherical,  white  colonies.  Upon  cooked  potato  a  thick,  viscous  layer 
is  formed  of  milk-white  color  ;  the  growth  upon  blood  serum  is  also 
abundant,  especially  in  the  incubating  oven.  This  micrococcus  is  a 
facultative  anaerobic. 

Pathogenesis. — Subcutaneous  inoculation  of  a  culture  of  this 
micrococcus  in  minute  quantity  is  fatal  to  white  mice  in  from  two  to 
six  days.  The  animals  remain  apparently  well  for  the  first  day  or 
two,  then  remain  quiet  and  somnolent  until  death  occurs.  The  cocci 
are  found  in  comparatively  small  numbers  in  the  blood  of  the  heart, 
but  are  more  numerous  in  the  spleen,  lungs,  liver,  and  kidneys,  from 
which  organs  beautiful  stained  preparations  may  be  made  show- 
ing the  tetrads  surrounded  by  their  transparent  capsule.  Common 
house  mice  and  field  mice  are,  for  the  most  part,  immune,  as  are  the 
rabbit  and  the  dog.  Guinea-pigs  sometimes  die  from  general  infec- 
tion, and  sometimes  a  local  abscess  is  the  only  result  of  a  subcutane- 
ous inoculation. 

19.     MICROCOCCUS  BOTRYOGENUS   (Rabe). 

Synonyms. — Micrococcus  of  "  myko-desmoids  "  of  the  horse;  Mi- 
crococcus askoformans  (Johne)  ;  Ascococcus  Johnei  (Cohn). 

First  described  by  Bellinger  (1870)  ;  morphological  characters  and 
location  in  the  diseased  tissues  described  by  Johne  (1884)  ;  biological 
characters  determined  by  Rabe  (1886). 

Is  found  in  certain  diffused  or  circumscribed  growths  in  the  con- 
nective tissue  of  horses — "  myko-desmoids." 

Morphology. — Micrococci,  having  a  diameter  of  1  to  1.5  /*,  usu- 
ally united  in  pairs. 

In  the  tissues  the  cocci  are  united  in  colonies  of  fifty  to  one  hun- 
dred /*  in  diameter,  and  these  are  associated  in  mulberry-like  masses 
visible  to  the  naked  eye.  The  separate  colonies  are  enclosed  in  a 
homogeneous,  transparent  envelope — as  in  Ascococcus  Billrothii. 
This  is  not  the  case,  however,  in  cultures  in  artificial  media. 

Stains  with  the  aniline  colors. 

Biological  Characters. — In  gelatin  plate  cultures  spherical, 
sharply  defined,  silver-gray  colonies  are  developed  ;  later  these  have 
a  yellowish  color  and  a  metallic  lustre,  and  the  plate  presents  the  ap- 
pearance of  being  powdered  with  grains  of  pollen.  It  gives  off  a 
peculiar  fruit-like  odor,  reminding  one  of  the  odor  of  strawberries. 
In  gelatin  stick  cultures  growth  occurs  along  the  line  of  puncture  as 
a  pale  grayish- white  line,  which  later  becomes  milk-white ;  an  air 


328  PATHOGENIC   MICROCOCCI 

bubble  forms  near  the  surface  of  the  gelatin  ;  very  slight  liquefac- 
tion occurs  in  the  immediate  vicinity  of  the  line  of  growth,  and  after 
a  time  the  grayish-white  thread  sinks  into  an  irregular  mass,  lying 
at  the  bottom  of  the  puncture.  Upon  nutrient  agar  scarcely  any  de- 
velopment occurs.  Upon  potato  the  growth  is  abundant,  in  the  form 
of  a  pale-yellow,  circular  layer,  and  the  culture  gives  off  the  peculiar 
odor  above  described. 

Pathogenesis. — When  inoculated  into  guinea-pigs  general  infec- 
tion and  death  result.  In  sheep  and  goats  it  produces  a  local  in- 
flammatory oedema  and  sometimes  necrosis  of  the  tissues.  In  horses 
inoculated  subcutaneously  an  inflammatory  oedema  first  occurs,  fol- 
lowed at  the  end  of  from  four  to  six  weeks  by  the  development  of  new 
growths  in  the  connective  tissue,  resembling  the  tumors  found  in 
cases  of  the  disease  in  the  animal  from  which  the  micrococcus  in 
question  was  first  cultivated.  These  tumors  contain  characteristic 
mulberry-like  conglomerations  of  colonies  made  up  of  the  coccus. 

20.     MICROCOCCUS   OF   MANFREDI. 

Synonym. — Micrococcus  of  progressive  granuloma  formation. 

Obtained  by  Manfredi  (1886)  from  the  sputum  of  two  cases  of 
croupous  pneumonia  following  measles. 

Morphology. — Oval  micrococci,  having  a  diameter  of  0.6  to  1.0  /* 
and  from  1.0  to  1.5  /*  in  length  ;  usually  associated  in  pairs,  and  oc- 
casionally in  short  chains  containing  three  or  four  elements. 

Stains  with  the  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — Aerobic ;  does  not  liquefy  gelatin. 
Upon  gelatin  plates  forms  small,  spherical  colonies,  at  first  grayish- 
white,  which  spread  out  upon  the  surface  as  thin,  transparent  plates, 
which  by  transmitted  light  have  a  bluish,  by  reflected  light  a  pearl- 
gray  color.  Later  these  become  thicker  and  have  a  pearly  lustre. 
Under  the  microscope  (forty  to  fifty  diameters)  the  colonies  are  seen 
to  be  slightly  granular  and  the  margins  have  an  irregular  outline. 
In  gelatin  stick  cultures  a  scanty  growth  occurs  along  the  line  of 
puncture,  and  a  rather  thin  and  limited  growth  about  the  point  of 
inoculation.  Upon  blood  serum  a  thin,  greenish-yellow  layer,  which 
has  irregular  margins  and  a  slightly  granular,  shining  surface,  is 
developed.  The  growth  upon  potato,  at  37°  C.,  is  scanty,  and  con- 
sists of  a  very  thin,  moist  layer,  which  has  a  yellowish  color  and  is 
slightly  granular.  Growth  occurs  in  favorable  media — bouillon, 
gelatin— at  temperatures  of  18°  to  48°  C.,  but  ceases  at  a  temperature 
of  48°  to  50°  C. 

Pathogenesis. — Pathogenic  for  dogs,  rabbits,  guinea-pigs,  mice, 
and  l.inls.  In  mammals  the  principal  pathological  appearance  re- 
sulting from  infection  consists  in  the  formation  of  "  granulation  tu- 


NOT   DESCRIBED   IN   SECTIONS  IV.    AND   V.  329 

mors  "  in  the  parenchymatous  organs.  These  vary  in  size  from  that 
of  a  millet  seed  to  that  of  a  pea,  and  undergo  caseation.  They  con- 
tain the  micrococcus  and  are  infectious.  Mammals  die  in  from  nine 
to  fifteen  days  ;  birds  in  from  one  to  three  or  four,  and  without  the 
formation  of  the  characteristic  granuloma,  but  with  general  infec- 
tion of  the  blood.  Cultures  which  have  been  kept  for  several  months 
retain  their  pathogenic  power. 

21.  MICROCOCCUS   OF   BOVINE   MASTITIS   (Kitt). 

Obtained  by  Kitt  (1885)  from  the  udder  of  cows  suffering  from  mastitis 
and  giving  milk  mixed  with  pus. 

Morphology. — Micrococci,  having  a  diameter  of  0.2  to  0.5  ju,  solitary, 
united  in  pairs,  in  irregular  groups,  and  occasionally  in  chains. 

Stains  with  the  aniline  colors. 

Biological  Characters. — Does  not  liquefy  gelatin.  Upon  gelatin  plates 
forms  spherical,  translucent,  glistening  colonies,  the  size  of  a  hemp  seed  to 
that  of  a  pin's  head ;  in  gelatin  stick  cultures  a  nail-shaped  growth  occurs, 
the  mass  at  the  point  of  puncture  being  opaque  and  of  a  white  color.  Upon 
potato,  colonies  are  quickly  developed  which  have  a  grayish -white  or  dirty 
yellow  color,  and  after  a  few  days  have  a  shining,  wax-like  appearance. 
Grows  rapidly  in  milk,  causing  an  acid  reaction ;  in  six  hours  in  the  incu- 
bating oven  the  milk  is  pervaded  by  the  micrococcus,  or  in  twelve  hours  at 
20°  C. 

Pathogenesis. — Injection  of  pure  cultures,  suspended  in  distilled  water, 
into  the  mammary  glands  of  cows,  produces  typical,  acute,  purulent  mas- 
titis (Kitt).  The  micrococcus  produced  the  same  result  after  having  been 
cultivated  in  artificial  media  for  a  year.  Subcutaneous  inoculations  in  cows, 
pigs,  guinea-pigs,  rabbits,  and  mice  were  without  result.  Injections  into 
the  mammary  gland  of  goats  were  also  without  effect. 

22.  MICROCOCCUS  OF  BOVINE  PNEUMONIA   (?). 

Isolated  by  Poels  and  Nolen  (1886)  from  the  lungs  of  cattle  suffering 
from  '"Limgenseuche"  (infectious  pleuro-pneumonia  of  cattle). 

Morphology. — Micrococci,  varying  considerably  in  size — average  dia- 
meter 0.9  #;  solitary,  in  pairs,  or  in  chains  containing  several  elements;  sur- 
rounded by  a  transparent  capsule,  which  stains  with  difficulty. 

Stains  with  all  the  aniline  colors,  and  with  difficulty  by  Gram's  method. 

Biological  Characters. — Does  not  liquefy  gelatin,  and  grows  like  the  ba- 
cillus of  Friedlander  in  gelatin  stick  cultures  (nail-shaped  growth).  In  gela- 
tin plates  the  colonies  are  spherical,  white,  and  have  a  very  faint  yellowish 
tinge.  Grows  more  rapidly  on  agar  in  the  incubating  oven,  and  upon  po- 
tato in  the  form  of  a  very  pale-yellowish  layer.  Is  destroyed  by  a  tempera- 
ture of  66°  C.  maintained  for  fifteen  minutes. 

Pathogenesis  — Pure  cultures  injected  into  the  lungs  of  dogs,  rabbits, 
and  guinea-pigs  are  said  to  give  rise  to  pneumonic  inflammation,  and  simi- 
lar results  were  obtained  by  injection  into  the  trachea  of  dogs  and  by  in- 
halation experiments.  Injection  of  a  pure  culture  into  the  lungs  of  a  cow 
caused  extensive  pneumonic  changes;  but  these  did  not  entirely  correspond 
with  the  appearances  found  in  the  lungs  of  cattle  suffering  from  infectious 
pneumonia.  Cattle  inoculated  with  a  pure  culture,  by  means  of  a  sterilized 
lancet,  did  not  fall  sick,  but  are  believed  by  Poels  and  Nolen  to  have  been 
protected  from  the  disease  by  such  inoculations. 

The  specific  relation  of  the  micrococcus  above  described  to  the  disease 
with  which  it  was  associated,  in  the  researches  of  the  authors  mentioned,  has 
not  been  established  by  subsequent  investigations. 
22 


330  PATHOGENIC  MICROCOCCI 

23.   STREPTOCOCCUS  SEPTICUS   (Fliigge). 

Found  by  Nicolaier  and  by  Guarneri  in  unclean  soil  during  researches 
made  in  Fliigge's  laboratory  in  Gottingen. 

Morphology. — Cannot  be  distinguished  from  Streptococcus  pyogenes,  but 
does  not  so  constantly  form  chains,  being  found  in  the  tissues  of  inoculated 
animals,  for  the  most  part  in  pairs. 

Biological  Characters.— Grows  more  slowly  than  Streptococcus  pyogenes ; 
in  gelatin  plates  very  minute  colonies  first  appear  at  the  end  of  three  or  four 
days,  or  along  the  line  of  puncture  in  gelatin  stick  cultures  after  five  or  six 
days.  Does  not  liquefy  gelatin. 

Pathogenesis. — Is  very  pathogenic  for  mice  and  for  rabbits,  causing  death 
from  general  infection  in  two  or  three  days. 

24.    STREPTOCOCCUS   BOMBYCIS. 

Synonym. — Microzyma  bombycis  (Bechamp). 

Found  in  the  bodies  of  infected  silkworms  suffering  from  la  flacherie 
(maladie  des  morts-plats).  Etiological  relation  established  by  Pasteur. 

Morphology. — Oval  cells,  not  exceeding  1.5  ju,  in  diameter,  in  pairs  or  in 
chains. 

Biological  Characters. — Not  determined  with  precision. 

Pathogenesis. — The  infected  silkworm  ceases  to  eat,  becomes  weak,  and 
dies.  Its  body  is  soft  and  diffluent,  and  at  the  end  of  twenty-four  to  forty- 
eight  hours  is  filled  with  a  dark-brown  fluid  and  with  gas. 

25.    NOSEMA  BOMBYCIS. 

Synonyms. — Micrococcus  ovatus;  Panhistophyton  ovatum. 

Found  in  the  blood  and  all  of  the  organs  of  silkworms  infected  with 
p4brine  (Fleckenkrankheit). 

First  observed  by  Cornalia.     Etiological  relation  established  by  Pasteur. 

Morphology. — Shining,  oval  cells,  three  to  four  #  long  and  two/*  broad; 
solitary,  in  pairs,  or  in  irregular  groups. 

Biological  Characters. — Not  determined  with  precision. 

Pathogenesis. — Dark  spots  appear  upon  the  skin  of  infected  silkworms, 
which  lose  their  appetite,  become  slender  and  feeble,  and  soon  die.  The 
oval  corpuscles  are  found  in  all  of  the  organs,  and  also  in  the  eggs  of 
butterflies  hatched  from  infected  larvae.  Some  authors  are  of  the  opinion 
that  the  oval  corpuscles  found  in  this  disease  do  not  belong  to  the  bacte- 
ria, but  to  an  entirely  different  class  of  microorganisms — the  Psorospermia 
(Metschnikoff). 

26.  MICROCOCCUS  OF  HEYDENREICH. 

Synonyms.— Micrococcus  of  Biskra  button— Fr.  "  clou  de  Biskra  ";  Ger. 
"Pendesche  Geschwur." 

Found  by  Heydenreich  (1888)  in  pus  and  serous  fluid  obtained  from  the 
tumors  and  ulcers  in  the  Oriental  skin  affection  known  as  Biskra  button. 

Morphology.—  Diplococci,  from  0.86  to  1  /*  in  length,  surrounded  by  a 
capsule ;  sometimes  associated  to  form  tetrads. 

,s'/a///.s  with  the  usual  aniline  colors. 

Biological  Characters .— An  aerobic,  liquefying  micrococcus  Grows  in 
the  usual  culture  media  at  the  room  temperature.  In  gelatin  stick  cultures, 
at 20°  C.,  at  the  end  of  fortv-eight  hours  growth  occurs  along  the  line  of 
puncture  in  the  form  of  small,  crowded  colonies,  which  produce  a  grayish- 
white  line;  upon  the  surface  a  thin,  circular  layer  of  a  yellowish-white 
color  is  developed.  At  the  end  of  three  to  four  days  liquefaction  commences 
near  the  surface,  where  a  funnel  is  formed  which  extends  until  about  the 
fourteenth  day,  when  the  gelatin  is  completely  liquefied.  Upon  the  surface 


NOT   DESCRIBED   IN   SECTIONS   IV.    AND   V.  331 

of  agar,  at  37°  C.,  a  grayish- white  or  yellowish  layer  is  formed  at  the  end  of 
twenty-four  hours,  which  has  a  varnish-like  lustre.  Upon  potato,  at  30°  to 
35°  C. ,  at  the  end  of  forty-eight  hours  a  white  or  yellow  layer  has  de- 
veloped. 

Pathogenesis. — According  to  Heydenreich,  inoculations  in  rabbits,  dogs, 
chickens,  horses,  and  sheep  cause  a  skin  affection  which  is  identical  with 
that  which  characterizes  Biskra  button  in  man.  When  rubbed  into  the 
healthy  skin  of  man  it  also  produces  the  development  of  abscesses. 


27.    MICROCOCCUS  OP  DEMME. 

Synonym. — Diplococcus  of  pemphigus  acutus  (Demme). 

Obtained  by  Demme  (1886)  from  the  contents  of  the  bullae  in  a  case  of 
pemphigus. 

Morphology.—  Micrococci  of  from  0.8  to  1.4  //  in  diameter;  usually  united 
in  pairs  resembling  the  "  gonococcus "  and  having  a  length  of  1.8to3/*, 
very  opaque  and  not  surrounded  by  a  capsule ;  usually  associated  in  irregu- 
lar masses. 

Biological  Characters. — Aerobic  micrococci.  Do  not  grow  at  the  room 
temperature.  Upon  agar  plates,  at  37°  C.,  at  the  end  of  thirty-six  to  forty- 
eight  hours  milk-white,  spherical  colonies,  which  project  above  the  surface, 
are  developed ;  later  club-shaped  outgrowths  form  around  the  periphery  of 
the  colony,  giving  it  the  appearance  of  a  rosette,  or  sometimes  of  a  bunch  of 
grapes.  At  the  end  of  two  weeks  the  surface  is  covered  with  smooih  projec- 
tions and  has  a  cream-like  color.  In  streak  cultures  upon  agar  a  similar 
growth  occurs  along  the  impfstrich,  having  club-like  projections  and  stalac- 
tite-like outgrowths.  Growth  also  occurs  upon  potato  at  a  temperature  of 
37°  C.  This  micrococcus  develops  slowly  in  the  incubating  oven,  and 
scarcely  any  growth  occurs  at  a  temperature  below  32°  C. 

Pathogenesis.  — The  injection  of  a  pure  culture  into  the  lungs  of  guinea- 
pigs  gave  rise  to  emaciation  and  debility  and  to  the  formation  of  foci  of 
broncho-pneumonia,  the  size  of  a  pea,  in  the  lungs. 

28.  STREPTOCOCCUS  OF  MANNEBERG. 

Obtained  by  Manneberg  (1888)  from  the  urine  in  acute  cases  of  Bright's 
disease. 

Morphology. — Micrococci,  aboutO.9//  in  diameter,  solitary,  in  pairs,  or 
in  chains  of  six  to  ten  elements.  Does  not  differ  in  morphology  from  Strep- 
tococcus pyogenes. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic  micro- 
coccus,  which  slowly  produces  a  viscid  softening  of  nutrient  gelatin.  Grows 
in  the  usual  culture  media  at  the  room  temperature.  In  gelatin  stick  cul- 
tures forms  a  white  stripe  along  the  line  of  puncture,  which  consists  of  small 
colonies.  At  the  end  of  three  or  four  weeks  a  funnel  is  formed  containing 
very  viscid  liquefied  gelatin,  and  at  the  same  time  brush-like  outgrowths  are 
seen  along  the  line  of  development.  Upon  the  surf  ace  of  agar  the  growth 
resembles  that  of  Streptococcus  pyogenes,  but  is  somewhat  more  abundant. 
IJpon potato,  at  37°  C.,  at  the  end  of  four  or  five  days  white,  drop-like  colo- 
nies are  developed  of  about  0.5  millimetre  in  diameter;  these  become  con- 
fluent and  form  a  slimy  layer.  Milk  becomes  strongly  acid  and  coagulates 
within  twelve  hours  when  inoculated  with  this  micrococcus. 

Pathoge nesis.— Subcutaneous  injection  of  0.75  to  1  cubic  centimetre 
causes  the  formation  of  a  local  abscess  in  dogs  and  rabbits.  Intravenous 
injections  produce  inflammatory  changes  in  the  kidneys ;  at  the  end  of  three 
or  four  days  the  urine  contains  red  blood  corpuscles,  renal  epithelium,  blood 
casts,  albumin,  and  streptococci. 


332  PATHOGENIC   MICROCOCCI 

29.  MICROCOCCUS  ENDOCARDITIDIS  RUGATUS  (Weichselbaum). 

Obtained  by  Weichselbaum  (1890)  from  the  affected  cardiac  valves  in  a 
fatal  case  of  ulcerative  endocarditis. 

Morphology. — Micrococci,  resembling-  the  staphylococci  of  pus  in  dimen- 
sions and  mode  of  grouping;  solitary,  in  pairs,  in  groups  of  four,  or  in  ir- 
regular masses. 

Biological  Characters. — An  aerobic  micrococcus.  Does  not  grow  at  the 
room  temperature.  Upon  agar  plates,  at  37°  C. ,  at  the  end  of  three  or  four 
days  the  superficial  colonies  consist  of  a  small,  brown,  central  mass  sur- 
rounded by  a  granular,  semi  transparent,  grayish  marginal  zone;  gradually 
they  attain  a  characteristic  wrinkled  appearance;  the  deep  colonies,  under  a 
low  power,  are  irregular,  finely  granular,  and  contain  a  large  central,  yel- 
lowish-brown nucleus  surrounded  by  a  narrow,  grayish -brown  peripheral 
zone.  In  agar  stick  cultures  small,  spherical  colonies  are  formed  upon  the 
surface,  which  become  confluent,  forming  a  grayish-white,  wrinkled  la}rer 
which  has  a  stearin-like  lustre  and  is  very  viscid ;  a  scanty  growth  occurs 
along  the  line  of  puncture.  Upon  potato,  at  37°  C  ,  a  scanty  development 
occurs  in  the  form  of  a  small,  dry,  pale-brown  mass.  Upon  blood  serum 
isolated  or  confluent,  colorless  colonies  are  formed  the  size  of  a  poppy  seed ; 
these  are  closely  adherent  to  the  surface  of  the  culture  medium. 

Pathogenesis. — When  injected  subcutaneously  into  the  ear  of  a  rabbit  it 
produces  tumefaction  and  redness;  in  guinea-pigs,  formation  of  pus.  When 
injected  into  the  circulation  of  dogs,  after  injury  to  the  aortic  valves,  an  en- 
docarditis is  developed. 

30.    MICROCOCCUS  OF  GANGRENOUS  MASTITIS  IN  SHEEP. 

Obtained  bjr  Nocard  (1887)  from  the  milk  of  sheep  suffering  from  gan- 
grenous mastitis  (rnal  de  pis  or  d'araignee),  a  fatal  disease  which  attacks 
especially  sheep  which  are  being  milked  for  the  manufacture  of  cheese,  at 
Roauefort  and  elsewhere  in  France. 

Morphology.—  Micrococci,  solitary,  in  pairs,  or  in  irregular  groups,  resem- 
bling the  staphylococci  of  pus  in  dimensions  and  arrangement. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  liquefy- 
ing micrococcus.  Grows  at  the  room  temperature  in  the  usual  culture  me- 
dia. Upon  gelatin  plates,  at  the  end  of  forty-eight  hours,  the  colonies  are 
spherical  and  white  in  color;  under  a  low  power  the  superficial  colonies  are 
circular  in  outline,  homogeneous,  and  brown  in  color;  they  are  surrounded 
by  a  semi -transparent  aureole  ;  liquefaction  around  the  superficial  colonies 
occurs  sooner  than  around  those  beneath  the  surface  of  the  gelatin.  In 
gelatin  stick  cultures,  at  18°  to  20°  C.,  on  the  second  day  liquefaction  of  the 
gelatin  commences  near  the  surface  ;  by  the  fifth  day  a  pouch  of  liquefied 
gelatin  has  formed,  which  has  the  shape  of  an  inverted  cone;  at  the  bottom 
of  this  an  abundant  deposit  of  micrococci  is  seen,  while  the  liquefied  gela- 
tin above  is  clouded  throughout.  In  agar  stick  cultures  development  oc- 
curs upon  the  surface  as  a  thick  white  layer,  which  gradually  extends 
over  the  entire  surface,  and  after  a  time  acquires  a  yellowish  tint;  develop- 
ment also  occurs  along  the  line  of  puncture.  Upon  potato  a  thin,  viscid, 
grayish  layer  is  slowlv  developed;  the  outline  is  irregular  and  the  edges 
Incker  than  the  central  portion  ;  the  central  portion  of  this  la  yc-r  gradually 
acquires  a  yellow  color,  while  the  periphery  remains  of  a  dirty-white  or 
grayish  color.  Blood  serum  is  liquefied  by  this  micrococcus. 

/  ''itliogenem—A.  few  drops  of  a  pure  culture  injected  subcutaneously  or 
ito  the  mammary  gland  of  sheep  cause  an  extensive  inflammatory  oedema 
and  the  death  of  the  animal  in  from  twenty-four  to  forty-eight  hours.  A 
<MI  .10  centimetre  injected  into  the  mammary  -land  of  a  goat  produced  no  re- 
'.'"'  h«»rs«.,  tho  calf,  tho  pi-.  1  In- cat,  chickens.  ;u.<l  guinea-piers  also  proved 
to  be  immune.  Subcutaneous  injections  in  rabbits  produce  an  extensive  ab- 
scess at  the  point  of  inoculation. 


NOT   DESCRIBED   IN   SECTIONS   IV.    AND    V. 
31.    STREPTOCOCCUS   OF   MASTITIS   IN   COWS. 


333 


Obtained  by  Nocard  and  Mollereau  (1887)  from  the  milk  of  cows  suffering- 
from  a  form  of  chronic  mastitis  (mammite  contagieuse) . 

Morphology.—  Spherical  or  oval  cocci,  a  little  less  than  one  /*  in  diameter, 
usually  united  in  long  chains. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  streptococcus.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  Develops  rapidly  in  milk  or  in  bouillon  at  a  temperature  of 
16°  to  30°  C.  The  milk  of  a  cow  suffering  from  the  form  of  mastitis  produced 
by  this  micrococcus,  when  drawn  with  proper  precautions  in  sterilized  test 
tubes,  at  the  end  of  twenty  four  hours  is  acid  in  reaction;  the  lower  two- 
thirds  of  the  tube  is  filled  with  an  opaque,  dirty- white,  homogeneous  deposit, 
and  above  this  is  an  opalescent,  serous  fluid  of  a  bluish  or  dirty-yellow  or 
slightly  reddish  color,  according  to  the  age  of  the  lesion.  A  drop  of  this 
milk  examined  under  the  microscope  shows  the  presence  of  the  streptococcus 
in  great  numbers.  The  addition  of  two  to  five  per  cent  of  glucose  or  of  gly- 


FIG.  99.— Streptococcus  of  mastitis  in  cows  (Nocard). 

cerin  to  bouillon  makes  it  a  more  favorable  culture  medium ;  the  reaction 
should  be  neutral  or  slightly  alkaline,  as  this  streptococcus  does  not  grow- 
readily  in  an  acid  medium,  although  it  produces  an  acid  reaction  in  media 
containing  sugar,  the  acid  formed  being  lactic.  In  gelatin  stick  cultures  the 
growth  upon  the  surface  is  scanty,  in  the  form  of  a  thin  pellicle  around  the 
point  of  puncture ;  along  the  line  of  inoculation  minute,  opaque,  granular 
colonies  are  developed,  which,  being  closely  crowded,  form  a  thick  line  with 
jagged  margins. 

In  agar  stick  cultures  the  growth  is  similar  but  more  abundant.  Upon 
the  surface  of  nutrient  gelatin,  agar,  or  blood  serum  a  large  number  of  mi- 
nute, spherical,  semi-transparent  colonies  are  developed  along  the  impfstrich ; 
these  have  a  bluish  tint  by  reflected  light ;  they  may  become  confluent,  form- 
ing a  thin  layer  with  well-defined  margins.  Upon  gelatin  plates,  at  16°  to 
18°  C.,  colonies  are  first  visible  at  the  end  of  two  or  three  days;  they  are 
spherical  and  slightly  granular,  at  first  transparent  and  later  of  a  pale-yellow 
color  by  transmitted  light,  which  gradually  becomes  brown.  At  the  end  of 
five  or  six  weeks  the  colonies  are  still  quite  small,  well  defined,  and  opaque. 

Pathogenesis. — Pure  cultures  injected  into  the  mammary  gland  of  cows 
and  goats  gave  rise  to  a  mastitis  resembling  in  its  development  that  from 


334  PATHOGENIC   MICROCOCCI 

which  fie  streptococcus  was  obtained  in  the  first  instance.  Injections  into 
the  cavity  of  the  abdomen  or  into  a  vein,  of  one  cubic  centimetre  of  a  pure 
culture,  gave  a  negative  result  in  dogs,  cats,  rabbits,  and  guinea-pigs. 

32.    DIPLOCOCCUS  OF  PNEUMONIA  IN  HORSES. 

Obtained  by  Schutz  (1887)  from  the  lungs  of  horses  affected  with  pneu- 
monia. 

Morphology. — Oval  cocci,  usually  in  pairs,  surrounded  by  a  homogene- 
qyus,  transparent  capsule. 

Does  not  stain  by  Gram's  method. 

Biological  Characters.— An  aerobic,  non-liquefying  micrococcus.  Grows 
at  the  room  temperature.  Upon  gelatin  plates  forms  small,  spherical,  white 
colonies. 

In  gelatin  stick  cultures  grows  along  the  line  of  puncture  as  small,  white, 
separate  colonies,  which  grow  larger  without  becoming  confluent.  Upon 
the  surface  of  agar  small  transparent  drops  are  developed  along  the  impf- 
strich. 

Pathogenesis.— The  injection  of  a  pure  culture  into  the  lung  of  a  horse 
produces  pneumonia  and  causes  its  death  in  eight  or  nine  days.  Pathogenic 
lor  rabbits,  guinea-pigs,  and  mice. 

33.   STREPTOCOCCUS  CORYZJE  CONTAGIOSvE  EQUORUM. 

Obtained  by  Schutz  (1888)  from  pus  from  the  lymphatic  glands  involved 
in  horses  suffering  from  the  disease  known  in  Germany  as  Druse  des 
Pferdes. 

Morphology. — Oval  cocci,  in  pairs,  in  chains  containing  three  or  four 
elements,  or  in  long  chaplets. 

Stains  with  the  usual  aniline  colors — very  intensely  with  Weigert's  or 
Ehrlich's  solution. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic  micrococ- 
cus. Grows  slowly  at  the  room  temperature,  more  rapidly  at  37°  C.  Upon 
gelatin  plates  at  the  end  of  three  to  five  days  minute  colonies  become  visible ; 
these  never  exceed  the  size  of  a  pin's  head.  In  gelatin  stick  cultures  growth 
upon  the  surface  is  scanty  or  absent;  along  the  line  of  puncture  minute 
colonies  are  developed  in  rows.  Upon  agar  plates,  at  37°  C.,  at  the  end  of 
twenty-four  hours  lentil-shaped  colonies  are  developed  the  size  of  a  pin's 
head;  under  a  low  power  the  superficial  colonies  are  seen  to  have  a  well-de- 
fined, opaque  nucleus  surrounded  by  a  grayish,  transparent  marginal  zone, 
which  represents  a  half-fluid,  slimy  growth  which  does  not  extend  after  the 
third  day  and  later  disappears  entirely;  the  deep  colonies  are  at  first  well- 
(iHined,  and  later  surrounded  by  wing-like  outgrowths.  Upon  blood  serum, 
;it  :\7'  C.,  yellowish,  transparent  drops  are  first  developed;  these  become  con- 
fluent and  form  a  viscid  and  tolerably  thick  layer;  this  later  becomes  dry 
and  iridescent. 

Pathogenesis. — Pathogenic  for  horses  and  for  mice,  producing  in  these 
animals  an  abscess  at  the  point  of  inoculation,  and  metastatic  abscesses  in 
the  neighboring  lymphatic  glands.  Not  pathogenic  for  rabbits,  guinea-pigs, 
or  pigeons. 

34.  H^MATOCOCCUS  BO  vis  (Babes). 

Obtained  by  Babes  (1889)  from  the  blood  and  various  organs  of  cattle 
which  had  died  of  an  epidemic  malady  (in  Roumania)  characterized  by  ha-im  >- 
globinuria.  The  cocci  are  found  in  the  blood  in  great  numbers,  for  the  most 
part  enclosed  in  the  red  corpuscles. 

Morphology. — Biscuit-shaped  cocci  united  in  pairs;  sometimes  oblong  in 
form,  isolated  01- united  in  groups;  the  free  cocci  are  surrounded  by  a  pale- 
yellowish,  shining  aureole  of  0.5  to  1  >u  in  diameter. 


NOT   DESCRIBED   IN   SECTIONS   IV.    AND    V.  335 

Stains  best  with  Loffler's  solution  of  methylene  blue ;  does  not  stain  by 
Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  micrococcus.  Grows  very  slowly  at  the  room  temperature— not 
below  20°  C.  In  the  incubating*  oven  grows  in  the  usual  culture  media.  In 
gelatin  stick  cultures  a  scanty  development  of  small,  white  colonies  occurs 
along  the  line  of  puncture.  Upon  the  surface  of  agar  small,  transparent 
drops  are  developed  along  the  impfstrich.  Upon  potato,  at  37°  C..  a  thin, 
broad,  yellowish,  shining  layer  is  developed  in  the  course  of  a  few  days— 
scarcely  visible.  Upon  blood  serum  small,  moist,  transparent  colonies  are 
developed. 

Pathogenesis. — Pathogenic  for  rabbits  and  rats,  which  die  in  from  six  to 
ten  days  after  inoculation  with  a  pure  culture;  the  spleen  is  found  to  be  en- 
larged, the  lungs  hyperasmic,  and  a  bloody  serum  is  found  in  the  cavity  of 
the  abdomen ;  the  cocci  are  present  in  the  blood  in  considerable  numbers, 
but  are  rarely  seen  in  the  red  corpuscles.  Inoculations  in  oxen,  horses, 
goats,  sheep,  guinea-pigs,  and  birds  were  without  effect. 

35.   MICROCOCCUS  GINGIV.E  PYOGENES. 

Obtained  by  Miller  (1889)  from  the  mouth  of  a  man  suffering  from  alveo- 
lar abscess. 

Morphology. — Large  cocci  of  irregular  dimensions,  solitary  or  in  pairs. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying micrococcus.  Grows  at  the  room  temperature  in  the  usual  media.  Upon 
gelatin  plates  it  forms  spherical,  well-defined  colonies,  which  under  a  low 
power  are  at  first  slightly  colored  and  later  opaque.  In  gelatin  stick  cultures 
an  abundant  development  occurs  both  upon  the  surface  and  along  the  line 
of  puncture.  Upon  the  surface  of  agar  a  tolerably  thick,  grayish  growth 
occurs  along  the  impfstrich,  which  has  a  purplish  tint  by  transmitted  light. 

Pathogenesis. — Subcutaneous  injections  in  mice  produce  a  local  abscess 
and  necrosis  of  the  skin,  followed  sometimes  by  death.  Injections  into  the 
cavity  of  the  abdomen  produced  peritonitis  and  death  in  from  twelve  to 
twenty-four  hours. 

36.    PSEUDODIPLOCOCCUS  PNEUMONIJE. 

Obtained  by  Bonome  (1888)  from  the  sero-fibrinous  exudate  in  an  autopsy 
of  an  individual  who  died  of  cerebro-spinal  meningitis. 

Morphology.— Oval  cocci,  in  pairs  or  in  chains  of  five  or  six  elements, 
often  surrounded  by  a  transparent  capsule;  not  to  be  distinguished  troi 
Micrococcus  pneumonias  crouposas. 

Stains  with  the  usual  aniline  colors  and  by  Gram  s  method. 

Biological  Characters.— An  aerobic,  non-liquefying  micrococcus.    Grows 
in  the  usual  culture  media  at  the  room  temperature  (Micrococcus  pneumonias 
crouposaedoes  not  grow  at  the  room  temperature).     In  gelatin  stick  cultures 
very  small  colonies  are  developed  along  the  line  of  puncture  at  the  end  o. 
twenty-four  to  twenty-eight  hours.     Upon  the  surface  of  agar  a  rattier 
scanty,  moist  layer  is  developed  along  the  impfstrich.     Upon  potato  a  tnm, 
scarcely  visible  layer  is  developed.     In  bouillon  the  development  is  abun- 
dant; the  culture  medium  acquires  a  very  acid  reaction  and  gives  ott  a  st 
odor  like  that  of  perspiration.  .         ,  .  , 

Pathoqenesis.—  Pathogenic  for  mice,  guinea-pigs,  and  rabbits,  in  WHICH 
animals  it  produces  fatal  septicaemia;  the  spleen  is  not  enlarged,  as  is  the 
case  in  animals  inoculated  with  Micrococcus  pneumonias  crouposae. 

37.    STREPTOCOCCUS  SEPTICUS  LIQUEFACIENS. 

Obtained  by  Babes  (1889)  from  the  blood  and  various  organs  of  a  child 
which  died  of  sapticsernia  following  scarlatina. 


336  PATHOGENIC  MICROCOCCI 

Morphology. — Micrococci,  about  0.3  to  0.4  /*  in  diameter,  in  pairs  or  in 
short  chains  in  which  the  elements  are  loosely  connected. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic,  liquefying  micrococcus.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  In  gelatin  stick  cultures 
at  the  end  of  twenty-four  hours  a  thin,  granular,  whitish  stripe  is  seen  along 
the  line  of  puncture,  while  the  surface  seems  somewhat  depressed;  later 
liquefaction  of  the  gelatin  occurs  in  funnel  form ;  the  liquefied  gelatin  is  but 
slightly  clouded,  and  upon  the  walls  of  the  funnel  peculiar,  flat,  white,  leaf- 
shaped,  jagged  colonies  are  seen.  Upon  the  surface  of  agar,  at  36°  C. ,  small, 
white,  thin,  shining,  transparent  colonies  are  developed,  which  may  attain 
a  diameter  of  two  to  three  millimetres.  Upon  blood  serum  a  scarcely  visible 
granular  layer  is  developed. 

Pathogenesis. — Subcutaneous  injections  in  mice  and  rabbits  produce 
local  inflammation  with  oedema,  and  death  occurs  in  about  six  days ;  the 
streptococci  are  found  in  large  numbers  in  the  effused  serum,  in  the  blood, 
and  in  the  spleen.  After  being  cultivated  for  some  time  in  artificial  media 
the  cultures  lose  their  pathogenic  power. 

38.   MICROCOCCUS  OF  KIRCHNER. 

Obtained  by  Kirchner  (1890)  from  the  bronchial  secretions  (in  sputum)  of 
patients  suffering  from  epidemic  influenza — soldiers  in  garrison  at  Hanover. 

Morphology. — Spherical  cocci,  usually  associated  in  pairs,  and  surrounded 
by  a  capsule.  Distinguished  from  Micrococcus  pneumoniae  crouposse  by  be- 
ing smaller,  quite  spherical,  and  the  elements  in  a  pair  being  more  widely 
separated  from  each  other.  Found  in  the  bronchial  secretion  in  scattered 
pairs,  or  associated  in  groups ;  occasionally  seen  in  chains. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological Characters.—  An  aerobic  micrococcus;  does  not  grow  in  flesh- 
peptone-gelatin  at  the  room  temperature.  Upon  agar  plates,  at  36°  C., 
small,  grayish- white,  transparent,  spherical  colonies  are  developed,  which 
later  form  round,  grayish-white  plaques.  In  agar  stick  cultures  an  abun- 
dant development  occurs  upon  the  surface,  extending  to  the  walls  of  the 
test  tube ;  growth  also  occurs  along  the  line  of  puncture. 

Pathogenesis. — Not  pathogenic  for  rabbits  or  for  white  mice.  A  guinea- 
pig  which  received  one  cubic  centimetre  of  a  bouillon  culture  in  the  pleural 
cavity  died  at  the  end  of  twenty -four  hours ;  the  spleen  was  not  enlarged ; 
lungs  hyperaemic ;  the  micrococci  were  found  in  the  blood  and  in  the  vari- 
ous organs.  Another  guinea-pig,  which  received  one  cubic  centimetre  of  a 
bouillon  culture  in  the  cavity  of  the  abdomen,  recovered  after  a  slight  indis- 
position. 

39.   MICKOCOCCUS  NO.   II.   OF  FISCHEL. 

Obtained  by  Fischel  (1891)  from  the  blood  of  two  cases  of  influenza. 

Morphology.—  Micrococci  of  from  1  to  1.25  n  in  diameter,  mostly  in 
pairs,  sometimes  in  chains. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  liquefy- 
ing micrococcus.  Grows  in  the  usual  culture  media  at  the  room  tempera 
ture.  Upon  gelatin  plates  minute  colonies,  visible  only  under  the  micro- 
scope, are  developed  at  the  end  of  three  days.  In  gelatin  stick  cultures  an 
abundant  milk-white  growth  occurs  along  the  line  of  puncture,  and  lique- 


- . -  -  potato,  at  37°  C.,  at  the  end  of  eight  days  „ 
thin,  shining  layer  of  a  yellowish-white  color,  and  about  one  centimetre 
broad  is  developed;  no  growth  upon  potato  at  the  room  temperature.  No 
growth  occurs  in  liquid  blood  serum  or  in  milk.  In  sterilized  water  this 
micrococcus  is  said  by  Fischel  to  lose  its  vitality  in  eight  hours 


NOT   DESCRIBED   IN   SECTIONS   IV.    AND   V.  337 

Pathogenesis. — Pathogenic  for  dogs  and  for  horses.  Intravenous  injec- 
tion of  three  to  four  cubic  centimetres  in  dogs  is  said  to  produce  symptoms 
resembling  those  of  distemper  in  this  animal,  viz.,  increased  temperature, 
catarrhal  conjunctivitis,  in  some  cases  keratitis,  and  in  some  a  mucous  dis- 
charge from  the  preputial  sac.  The  micrococcus  was  not  found  «in  the  blood 
of  the  dogs  inoculated  by  intravenous  injection,  later  than  the  fourth  day. 


40.    STREPTOCOCCUS   OF  BONOME. 

Obtained  by  Bonome  (1890)  from  the  exudations  of  the  cerebro-spinal 
meninges  and  from  haemorrhagic  extravasations  in  the  lungs  in  cases  of 
epidemic  cerebro-spinal  meningitis. 

This  streptococcus  is  said  by  Bonome  to  be  distinguished  from  previously 
known  streptococci  by  the  following  characters:  It  does  riot  grow  readily 
in  artificial  culture  media,  and  soon  loses  its  pathogenic  power  when  pre- 
served in  a  desiccated  condition  or  cultivated  through  a  few  successive  gene- 
rations. It  differs  from  the  "  pneumococcus  "  and  "  meningococcus  "  by 
the  ball- shaped  appearance  of  its  colonies  on  agar  plates,  and  in  the  fact 
that  it  does  not  grow  upon,  blood  serum ;  also  by  the  difficulty  experienced 
in  carrying  it  through  five  or  six  generations  in  artificial  media. 

Pathogenesis.— In  white  mice  and  in  rabbits  a  fibrinous  inflammation 
and  death  result  from  inoculations  with  a  pure  culture,  the  symptoms  re- 
sembling those  produced  by  similar  inoculations  with  Micrococcus  pneumo- 
niae  crouposse.  It  does  not  produce  septicaemia  in  white  mice,  but  in  rabbits 
the  cocci  are  found  in  the  blood  in  chains  surrounded  by  a  capsule.  In 
guinea-pigs  and  dogs  a  local  fibrinous  inflammation  results  from  inocula- 
tions, and  the  streptococcus  is  found  in  the  gelatinous  exudate  at  the  point  of 
inoculation.  It  is  distinguished  from  the  streptococcus  of  erysipelas  by  its 
failure  to  grow  in  gelatin  or  in  blood  serum,  and  by  the  appearance  of  its 
colonies  on  agar  plates. 

41.      MICROCOCCUS  OF  ALMQUIST. 

Obtained  by  Almquist  (1891)  from  the  bullse  of  pemphigus  neonatorum, 
in  nine  children  suffering  from  this  disease  during  an  epidemic  which  oc- 
curred at  Goteborg. 

Morphology. — Micrococci  from  0.5  to  1  ju.  in  diameter,  usually  in  pairs. 

Stains  readily  with  the  aniline  colors. 

Biological  Characters. — An  aerobic,  liquefying,  chromogenic  micro- 
coccus.  Closely  resembles  Staphylococcus  pyogenes  aureus  in  its  morpho- 
logy and  growth  in  culture  media.  Produces  a  similar  golden-yellow  pig- 
ment. 

Pathogenesis. — According  to  Almquist,  this  micrococcus  is  distinguished 
from  Staphylococcus  pyogenes  aureus  by  its  specific  pathogenic  power.  Two 
inoculations  made  from  a  pure  culture,  by  means  of  a  lancet,  upon  his  own 
arm  gave  rise  to  a  development  of  bullse  like  those  of  pemphigus.  The 
process  showed  no  disposition  to  extend  deeper,  but  the  epidermis  was  raised 
by  a  collection  of  fluid  which  was  at  first  transparent  and  later  had  a  milky 
opacity.  From  the  contents  of  these  bullae  the  same  coccus  was  obtained  in 
pure  cultures. 

42.    STAPHYLOCOCCUS   PYOSEPTICUS. 

Obtained  by  Hericourt  and  Richet  (1888)  from  an  abscess  in  the  skin  of  a 
dog. 

In  its  morphology  and  biological  characters  this  micrococcus  closely  re- 
sembles Staphylococcus  pyogenes  albus,  and  it  is  probably  a  pathogenic  va- 
riety of  this  common  species.  But  the  experiments  made  by  the  authors 
referred  to  show  it  to  be  decidedly  more  pathogenic  for  rabbits.  Subcutane- 
ous injections  of  a  drop  or  two  of  a  pure  culture  caused  an  extensive  inflam- 
matory oedema,  and  death  in  from  twelve  to  twenty-four  hours. 


338       PATHOGENIC   MICROCOCCI   NOT   HERETOFORE  DESCRIBED. 

43.    STREPTOCOCCUS   PERNICIOSUS   PSITTACORUM. 

Micrococcus  of  gray  parrot  disease.  Eberth  and  Wolff  have  described 
an  infectious  disease  of  gray  parrots,  which  is  said  to  be  extremely  fatal 
among  the  imported  birds.  The  disease  is  characterized  by  the  formation  of 
nodules  upon  the  surface  and  in  the  interior  of  various  organs,  and  especially 
in  the  liver.  Micrococci  of  medium  size  are  found  in  these  nodules  and  in 
blood  from  the  heart;  these  are  sometimes  in  chains.  Microscopic  examina- 
tion of  stained  sections  shows  that  these  cocci  are  directly  related  to  the  tis- 
sue necrosis  which  characterizes  the  disease.  But  the  micrococcus  has  not 
been  cultivated  and  its  biological  characters  are  undetermined. 

44.   MICROCOCCUS  OF  FORBES. 

Forbes  (1886)  has  studied  an  infectious  disease  of  cabbage  caterpillars 
(Pieris  rapse),  which  appears  to  be  due  to  a  micrococcus  found  by  him  in 
large  numbers  in  the  bodies  of  the  infected  larvae.  This  micrococcus,  which 
resembles  the  common  staphylococci  in  form,  was  cultivated  in  liquid  media 
and  successful  inoculation  experiments  were  made. 

44A.    STREPTOCOCCUS  AGALACTI^E  CONTAGIOS^. 

Obtained  by  Adametz  (1894)  from  the  milk  of  cows  suffering  from  mas- 
titis (Gelben  Gait).  According  to  Adametz  all  of  the  streptococci  which 
have  been  described  by  different  investigators  (Kitt,  Nocard  and  Mollereau, 
Guillebeau,  and  others)  are  probably  varieties  of  a  single  species. 

Morphology. — Spherical  cocci  in  short  chains — 1  p  in  diameter. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
efying  streptococcus. 

Jpon  gelatin  plates  forms  flat,  transparent,  white  or  bluish-white, 
slimy  colonies,  having  a  slight  pearly  lustre  and  an  irregular  outline.  In 
nutrient  gelatin  containing  five  per  cent  of  milk  sugar  the  colonies,  at  the 
end  of  eight  days,  have  a  diameter  of  0.85  to  1  millimetre;  they  are  milk- 
white  and  of  a  semi-fluid,  slimy  consistence. 

Upon  agar  plates  the  deep  colonies  are  jmnctiform  and  white  in  color- 
under  a  low  power  they  are  seen  to  have  an  irregular  dentate  contour  and  a 
brownish  color;  the  superficial  colonies  gradually  assume  the  appearance 
of  transparent,  flat  drops  having  a  diameter  of  0.5  to  0.7  millimetre.  In 
sterilized  milk  fermentation  occurs,  at  37°  C.,  in  from  twenty  to  twenty-four 
hours;  some  hours  later  the  casein  is  precipitated,  fine  gas  bubbles  are  seen 
in  the  lower  part  of  the  fluid  and  a  foam  upon  the  surface;  the  reaction  is 
acid  and  the  casein  is  not  peptonized.  The  power  of  producing  acid  and  gas 
is  diminished  or  lost  after  a  few  successive  cultures  have  been  made. 

Streptococcus  mastitis  sporadice  (Guillebeau)  is  said  by  Adametz  to  be 
distinguished  from  the  streptococcus  above  described  (No.  44A)  by  being 
smaller — 0.5  p.  in  diameter — and  by  the  fact  that  the  cultures  do  not  lose  the 
power  of  producing  fermentation  in  milk. 


liqueh 
Upt 


VII. 

THE  BACILLUS  OF  ANTHRAX. 
[Fr.,  CHARBON;    Ger.,  MILZBRAND.] 

ANTHRAX  is  a  fatal  infectious  disease  which  prevails  extensively 
among  sheep  and  cattle  in  various  parts  of  the  world,  causing  heavy 
losses.  In  Siberia  it  constitutes  a  veritable  scourge  and  is  known 
there  as  the  Siberian  plague  ;  it  also  prevails  to  a  considerable  extent 
in  portions  of  France,  Hungary,  Germany,  Persia,  and  India,  and 
local  epidemics  have  occasionally  occurred  in  England,  where  it  is 
known  under  the  name  of  splenic  fever.  It  does  not  prevail  in  the 
United  States.  In  infected  districts  the  greatest  losses  are  incurred 
during  the  summer  season. 

In  man  accidental  inoculation  may  occur  among  those  who  come 
in  contact  with  infected  animals,  and  especially  during  the  removal  of 
the  skin  and  cutting -up  of  dead  animals,  when  there  is  any  cut  or 
abrasion  upon  the  hands.  A  malignant  pustule  is  developed  as  the 
result  of  such  inoculation,  but,  as  a  rule,  general  infection  does 
not  occur,  as  is  the  case  when  inoculations  are  made  into  the  more 
susceptible  lower  animals — rabbit,  guinea-pig,  mouse.  Those  who 
handle  the  hair,  hides,  or  wool  of  infected  animals  are  also  liable  to 
contract  the  disease  by  inoculation  through  opsn  wounds,  or  by  the 
inhalation  of  dust  containing  spores  of  the  anthrax  bacillus.  Cases 
of  pulmonic  anthrax,  known  formerly  in  England  as  "wool-sorters' 
disease/'  have  been  occasionally  observed  in  England  and  in  Ger- 
many, and  are  now  recognized  as  being  due  to  infection  through  the 
lungs  in  the  manner  indicated. 

The  French  physician  Davaine,  who  had  observed  the  anthrax 
bacillus  in  the  blood  of  infected  animals  in  1850,  communicated  to 
the  French  Academy  of  Sciences  the  results  of  his  inoculation  experi- 
ments in  1863  and  1864,  and  asserted  the  etiological  relation  of  the 
bacillus  to  the  disease  with  which  his  investigations  showed  it  to  be 
constantly  associated.  This  conclusion  was  vigorously  contested  by 
conservative  opponents,  but  has  been  fully  established  by  subsequent 
investigations,  which  show  that  the  bacillus,  in  pure  cultures,  induces 


840 


THE   BACILLUS   OF  ANTHRAX. 


anthrax  in  susceptible  animals  as  certainly  as  does  the  blood  of  an 
animal  recently  dead  from  the  disease. 

Owing  to  the  fact  that  this  was  the  first  pathogenic  bacillus  cul- 
tivated in  artificial  media,  and  to  the  facility  with  which  it  grows  in 
various  media,  it  has  served  more  than  any  other  microorganism  for 
researches  relating  to  a  variety  of  questions  in  pathology,  general 
biology,  and  public  hygiene,  some  of  which  are  discussed  in  other 
sections  of  this  volume. 

45.     BACILLUS  ANTHRACIS. 

Synonyms. — Milzbrandbacillus,  Ger.;  Bacteridie  du  charbon,  Fr. 

First  observed  in  the  blood  of  infected  animals  by  Pollender  (1849) 

and  by  Davaine  (1850).     Etiological  relation  affirmed  by  Davaine 


PIG.  lOO.-Bacillus  anthracis,  from  a  culture,  showing  development  of  long  threads  in  convo- 
luted bundles,  x  300.  (Klein.) 

(1863),  and  established  by  the  inoculation  of  pure  cultures  by  Pasteur 
(1879)  and  by  many  other  investigators. 

Morphology.—  Rod-shaped  bacteria  having  a  breadth  of  1  to 
1.25  /*,  and  5  to  20  ^  in  length;  or,  in  suitable  culture  media,  growing 
out  into  long,  flexible  filaments,  which  are  frequently  united  in 
twisted,  cord-like  bundles.  These  filaments  in  hanging-drop  cul- 
tures, before  the  development  of  spores,  appear  to  be  homogeneous  ; 
or  the  protoplasm  is  clouded  and  granular,  but  without  distinct  seg- 
mentation. But  in  stained  preparations  the  filaments  are  seen  to  be 
made  up  of  a  series  of  rectangular,  deeply  stained  segments.  In 
hanging-drop  cultures  the  ends  of  the  rods  appear  rounded,  but  in 
stained  preparations  from  the  blood  of  an  infected  animal  they  are 
seen  to  present  a  slight  concavity,  and  a  lenticular  interspace  is 
formed  where  two  rods  come  together.  The  diameter  of  the  rods 


THE   BACILLUS   OF   ANTHRAX. 


341 


varies  considerably  in  different  culture  media ;  and  in  old  cultures 
irregular  forms  are  frequently  seen — "  involution  forms." 

Under  favorable  conditions  endogenous  spores  are  developed  in 
the  long  filaments  which  grow  out  in  artificial  culture  media. 
These  first  appear  as  refractive  granules  distributed  at  regular  inter- 
vals in  the  segments  of  the  protoplasm,  which  gradually  disappear 
as  the  spores  are  developed ;  and  these  are  left  as  oval,  highly  re- 
fractive bodies,  held  together  in  a  linear  series  by  the  cellular  enve- 
lope, and  subsequently  set  free  by  its  dissolution.  The  germination 
of  these  reproductive  bodies  results  in  the  formation  of  rods  and 
spore-bearing  filaments  like  those  heretofore  described.  In  this  pro- 
cess the  spore  is  first  observed  to 
lose  its  brilliancy,  from  the  ab- 
sorption of  moisture,  a  promi- 
nence occurs  at  one  end  of  the 
oval  body,  and  soon  the  external 
envelope  —  exosporium — is  rup- 
tured, permitting  the  softened 
protoplasmic  contents  enclosed 
in  the  internal  spore  membrane 
— endosporium — to  escape  as  a 
short  rod,  to  which  the  empty 
exosporium  sometimes  remains 
attached. 

The  anthrax  bacillus  stains 
readily  with  the  aniline  colors 
and  also  by  Gram's  method, 
when  not  left  too  long  in  the 
decolorizing  iodine  solution. 
Loffler's  solution  of  methylene 
blue  is  an  especially  good  stain- 
ing fluid  for  this  as  well  as  for  many  other  bacilli.  Bismarck  brown 
is  well  adapted  for  specimens  which  are  to  be  photographed,  and  also 
for  permanent  preparations,  as  it  is  less  liable  to  fade  than  the  blue 
and  some  other  aniline  colors. 

Biological  Characters. — The  anthrax  bacillus  is  aerobic,  but 
not  strictly  so,  as  is  shown  by  the  fact  that  it  grows  to  the  bottom  of 
the  line  of  puncture  in  stick  cultures  in  solid  media.  It  is  non-mo- 
tile, and  is  distinguished  by  this  character  from  certain  common 
bacilli  resembling  it  in  morphology — Bacillus  subtilis — which  were 
frequently  confounded  with  it  in  the  earlier  days  of  bacteriological 
investigation. 

The  anthrax  bacillus  grows  in  a  variety  of  nutrient  media  at  a 


FIG.  101.— Bacillus  anthracis,  from  a  culture, 
showing  formation  of  spores,    x  1,000.    (Klein.) 


342 


THE   BACILLUS   OF   ANTHRAX. 


temperature  of  20°  to  38°  C.     Development  ceases  at  temperatures 
below  12°  C.  or  above  45°  C. 

This  bacillus  grows  best  in  neutral  or  slightly  alkaline  media,  and 
its  development  is  arrested  by  a  decidedly  acid  reaction  of  the  cul- 
ture medium.  It  may  be  cultivated  in  infusions  of  flesh  or  of  vari- 
ous vegetables,  in  diluted  urine,  in  milk,  etc. 

In  gelatin  plate  cultures  small,  white,  opaque  colonies  are  devel- 
oped in  from  twenty-four  to  thirty-six  hours,  which  under  the  micro- 
scope are  seen  to  be  somewhat  irregular  in  outline  and  of  a  greenish 
tint ;  later  the  colonies  spread  out  upon  the  surface  of  the  gelatin, 
and  the  darker  central  portion  is  surrounded  by  a  brownish  mass  of 
wavy  filaments,  which  are  associated  in  tangled  bundles.  Mycelial- 

like  outgrowths  from  the  periphery  of 
the  colony  may  often  be  seen  extending 
into  the  surrounding  gelatin.  At  the 
end  of  two  or  three  days  liquefaction  of 
the  gelatin  commences,  and  the  colony 
is  soon  surrounded  by  the  liquefied  me- 
dium, upon  the  surface  of  which  it  floats 
as  an  irregular  white  pellicle.  In  gela- 
tin stick  cultures  growth  occurs  all 
along  the  line  of  puncture  as  a  white  cen- 
tral thread,  from  which  lateral  thread- 
like ramifications  extend  into  the  culture 
medium.  At  the  end  of  two  or  three 
days  liquefaction  of  the  culture  medium 
commences  near  the  surface,  where  the 
development  has  been  most  abundant. 
At  first  a  pasty,  white  mass  is  formed, 
but  as  liquefaction  progresses  the  upper 
part  of  the  liquefied  gelatin  becomes 
transparent  from  the  subsidence  of  the 
motionless  bacilli,  and  these  are  seen 
upon  the  surface  of  the  non-liquefied 
portion  of  the  medium  in  the  form  of 

cloudy,  white  masses,  while  below  the  line  of  liquefaction  the  charac- 
teristic branching  growth  may  still  be  seen  along  the  line  of  puncture. 
In  agar  plate  cultures,  in  the  incubating  oven  at  35°  to  37°  C., 
colonies  are  developed  within  twenty-four  hours,  which  under  the 
microscope  are  seen  to  be  made  up  of  interlaced  filaments  and  are 
very  characteristic  and  beautiful.  Upon  the  surface  of  nutrient  agar 
a  grayish-white  layer  is  formed,  which  may  be  removed  in  ribbon-like 
strips  ;  and  in  stick  cultures  in  this  medium  a  branching  growth  is 
seen,  like  that  in  gelatin,  but  without  liquefaction.  The  addition  of 


Fia.  102.— Culture  of  Bacillus  an- 
thracis  In  nutrient  gelatin  :  a,  end 
of  four  days ;  6,  end  of  eight  days. 
(Baumgarten.) 


THE   BACILLUS   OP   ANTHRAX.  343 

a  small  quantity  of  agar  to  a  gelatin  medium  prevents  liquefaction 
of  the  gelatin  (Fliigge). 

Upon  blood  serum  a  rather  thick,  white  layer  is  formed  and 
liquefaction  slowly  occurs. 

Upon  potato  the  growth  is  abundant  as  a  rather  dry,  grayish- 
white  layer,  of  limited  extent,  having  a  somewhat  rough  surface  and 
irregular  margins. 

Spores  are  formed  only  in  the  free  presence  of  oxygen,  as  in  sur- 
face cultures  upon  potato  or  nutrient  agar,  or  in  shallow  cultures  in 
liquid  media,  and  at  a  temperature  of  20°  to  35°  C.  They  are  not 
formed  during  the  development  of  the  bacilli  in  the  bodies  of  living 


FIG.  103.— Colonies  of  Bacillus  anthracis  upon  gelatin  plates :  a,  at  end  of  twenty-four  hours; 
6,  at  end  of  forty -eight  hours,    x  80.    (Flugge.) 

animals,  but  after  the  death  of  the  animal  the  bacillus  continues  to 
multiply  for  a  time,  and  spores  may  be  formed  where  the  fluids 
containing  it  come  in  contact  with  the  air — as,  for  example,  in 
bloody  discharges  from  the  nostrils  or  from  the  bowels  of  the  dead 
animal. 

Varieties  incapable  of  spore  production  have  been  produced  arti- 
ficially, by  several  bacteriologists,  by  cultivating  the  bacillus  under 
unfavorable  conditions.  Roux  was  able  to  produce  a  sporeless  va- 
riety by  successive  cultivation  in  media  containing  a  small  quantity 
of  carbolic  acid — 1  : 1,000. 

Varieties  differing  in  their  pathogenic  power  may  also  be  pro- 
duced by  cultivation  under  unfavorable  conditions.  Thus  Pasteur 


344  THE  BACILLUS  OF  ANTHRAX. 

produced  an  "  attenuated  virus"  by  keeping  his  cultures  for  a  con- 
siderable time  before  replanting  them  upon  fresh  soil,  and  supposed 
the  effect  was  due  to  the  action  of  atmospheric  oxygen.  It  seems 
probable  that  it  was  rather  due  to  the  deleterious  action  of  its  own 
products  of  growth  present  in  the  culture  media.  It  has  been 
shown  by  Chamberlain  and  Roux  that  cultivation  in  the  presence 
of  certain  chemical  substances  added  to  the  culture  medium— e.  g. , 
bichromate  of  potassium  0.01  per  cent — causes  an  attenuation  of 
virulence.  The  same  result  occurs  when  cultures  are  subjected  to  a 
temperature  a  little  below  that  which  is  fatal  to  the  bacillus — 50°  C. 
for  eighteen  minutes  (Chauveau);  42.5°  C.  for  two  or  three  weeks 
(Koch).  Attenuation  of  pathogenic  virulence  is  also  effected  by  cul- 
tivation in  the  body  of  a  non-susceptible  animal,  like  the  frog  (Lu- 
barsch,  Petruschky);  or  in  the  blood  of  a  rat  (Behring);  by  exposure 
to  sunlight  (Arloing);  and  by  compressed  air  (Chauveau). 

Anthrax  spores  may  be  preserved  in  a  desiccated  condition  for 
years  without  losing  their  vitality  or  pathogenic  virulence  when  in- 
oculated into  susceptible  animals.  They  also  resist  a  comparatively 
high  temperature.  Thus  Koch  and  Wolffhiigel  found  that  dry  spores 
exposed  in  dry  air  required  a  temperature  of  140°  C.,  maintained  for 
three  hours,  to  insure  their  destruction.  But  spores  suspended  in  a 
liquid  are  destroyed  in  four  minutes  by  the  boiling  temperature, 
100°  C.  (writer's  determination). 

The  bacilli,  in  the  absence  of  spores,  according  to  Chauveau,  are 
destroyed  in  ten  minutes  by  a  temperature  of  54°  C. 

For  the  action  of  various  antiseptic  and  germicidal  agents  upon 
this  bacillus  we  must  refer  to  the  sections  especially  devoted  to  this 
subject  (Part  Second). 

Toussaint,  by  injecting  filtered  anthrax  blood  into  animals,  obtained 
evidence  that  it  contained  some  toxic  substance  which  in  his  experi- 
ments gave  rise  to  local  inflammation  without  any  noticeable  general 
symptoms.  More  recent  investigations  show  that  a  poisonous  sub- 
stance is  formed  during  the  growth  of  the  anthrax  bacillus,  and  that 
cultures  containing  this  toxin,  from  which  the  bacilli  have  been  re- 
moved by  filtration  through  porcelain,  produce  immunity  when  in- 
jected into  susceptible  animals,  similar  to  that  resulting  from  inocu- 
lations with  an  attenuated  virus.  It  is  probable  that  the  pathogenic 
power  of  the  anthrax  bacillus  depends  largely  upon  the  presence  of 
tliis  toxin,  and  that  the  essential  difference  between  virulent  and 
attenuated  varieties  depends  upon  the  more  abundant  production  of 
this  toxic  substance  by  the  former.  It  has  also  been  shown  that 
virulent  cultures  produce  a  larger  quantity  of  acid  than  those  which 
have  been  attenuated  by  any  of  the  agencies  above  mentioned 
(IVhring). 


THE   BACILLUS   OF   ANTHRAX.  345 

Martin  (1890)  has  studied  the  chemical  products  in  filtered  cul- 
tures of  the  anthrax  bacillus  and  obtained  the  following  results: 

1.  Protoalbumose,  deuteroalbumose,  and  a  trace  of  peptone.     The 
mixed  albumoses  were  found  not  to  be  poisonous  except  in  consider- 
able   doses — 0.3  gramme  injected  subcutaneously  killed  a  mouse 
weighing  twenty-two    grammes;    smaller  doses  produced  a  local 
oedema.     A  fatal  dose  caused  extensive  cedema,  coma,  and  death  in 
twenty-four  hours ;  the  spleen  was  sometimes  enlarged.     Boiling 
neutralizes  to  a  considerable  extent  the  toxic  power. 

2.  An  alkaloid,  soluble  in  water  and  in  alcohol,  but  insoluble  in 
benzol,  chloroform,  or  ether.     The  solutions  have  a  strongly  alkaline 
reaction,  and  crystalline  salts  are  formed  with  various  acids.     This 
alkaloid  is  somewhat  volatile,  and  when  exposed  to  light  loses  to  a 
considerable  extent  its  toxic  properties.     It  produces  symptoms  simi- 
lar to  those  resulting  from  inoculations  with  the  albumoses,  but  is 
more  toxic  and  more  prompt  in  its  action.     The  animal  quickly  falls 
into  a  state  of  coma ;  there  is  extensive  oedema  about  the  point  of 
inoculation,  and  the  spleen  is  usually  enlarged.    The  fatal  dose  for  a 
mouse  weighing  twenty- two  grammes  is  from  0.1  to  0.15  gramme  ; 
death  occurs  within  two  or  three  hours. 

3.  In  addition  to  these  toxic  substances  small  quantities  of  leucin 
and  of  tyrosin  were  found  in  the  filtered  cultures. 

Petermann  (1892)  has  made  a  series  of  experiments  with  filtered 
cultures  of  the  anthrax  bacillus  which  led  him  to  the  conclusion  that 
"  large  quantities  of  a  culture  in  serum  from  the  ox,  filtered  through 
porcelain,  injected  into  the  veins  of  a  susceptible  animal,  have  a  pre- 
ventive action ;  but  the  immunity  thus  conferred  is  transitory,  not 
lasting  longer  than  a  month  or  two." 

Pathogenesis. — The  anthrax  bacillus  is  pathogenic  for  cattle, 
sheep,  horses,  rabbits,  guinea-pigs,  and  mice.  White  rats,  dogs,  and 
frogs  are  immune,  as  is  also  the  Algerian  race  of  sheep.  The  spar- 
row is  susceptible  to  general  infection,  but  chickens,  under  normal 
conditions,  are  not.  Young  animals  are,  as  a  rule,  more  susceptible 
than  adults  of  the  same  species.  Man  does  not  belong  among  the 
most  susceptible  animals,  but  is  subject  to  local  infection  as  a  result 
of  accidental  inoculation — malignant  pustule — and  to  pulmonic  an- 
thrax from  breathing  air,  containing  spores  of  the  anthrax  bacillus, 
during  the  sorting  of  wool  or  hair  from  infected  animals.  In  animals 
which  have  a  partial  immunity,  natural  or  acquired,  as  a  result  of 
inoculations  with  attenuated  virus,  the  subcutaneous  introduction  of 
virulent  cultures  may  give  rise  to  a  limited  local  inflammatory  pro- 
cess, with  effusion  of  bloody  serum  in  which  the  bacillus  is  found  in 
considerable  numbers  ;  but  the  blood  is  not  invaded,  and  the  animal, 
after  some  slight  symptoms  of  indisposition,  recovers.  In  susceptible 
23 


346 


THE  BACILLUS  OF  ANTHRAX. 


animals  injections  beneath  the  skin  or  into  a  vein  give  rise  to  general 
infection,  and  the  bacilli  multiply  rapidly  in  the  circulating  fluid. 
Death  occurs  in  mice  within  twenty-four  hours,  and  in  rabbits,  as  a 
rule,  in  less  than  forty-eight  hours.  The  blood  of  the  heart  and 
large  vessels  may  be  found,  in  an  autopsy  made  immediately  after 
death,  to  contain  comparatively  few  bacilli  ;  but  in  the  capillaries  of 
the  various  organs,  and  especially  in  the  greatly  enlarged  spleen,  in 
the  liver,  the  kidneys,  and  the  lungs,  they  will  be  found  in  great 
numbers,  and  well-stained  sections  of  these  organs  will  give  an  as- 
tonishing picture  under  the  microscope,  which  the  student  should  not 
fail  to  see  in  preparations  made  by  himself.  The  capillaries  in  many 
places  will  be  found  stuffed  full  of  bacilli  ;  or  they  may  even  be  rup- 
tured as  a  result  of  the  distention,  and  the  bacilli,  together  with 


Fio.  104.— Bacillus  anthracis  in  liver  of  mouse,    x  700.    (Flugge.) 

escaped  blood  corpuscles,  will  be  seen  in  the  surrounding  tissues.  In 
the  kidneys  the  glomeruli,  especially,  appear  as  if  injected  with  col- 
ored threads,  and  by  rupture  these  may  find  their  way  into  the  urini- 
ferous  tubules. 

These  appearances  and  the  general  symptoms  indicate  that  the 
disease  produced  by  the  introduction  of  this  bacillus  into  the  bodies  of 
susceptible  animals  is  a  genuine  septicaemia.  As  in  other  forms  of 
septicaemia,  the  spleen  is  found  to  be  greatly  enlarged  ;  it  has  a  dark 
color  and  is  soft  and  friable.  With  this  exception  the  organs  pre- 
sent no  notable  changes,  although  the  liver  is  apt  to  be  somewhat 
enlarged.  In  the  guinea-pig  an  extensive  inflammatory  oedema,  ex- 
tending from  the  point  of  inoculation  to  the  most  dependent  parts  of 
the  body,  is  developed ;  the  subcutaneous  connective  tissue  is  infil- 
trated with  bloody  serum  and  has  a  gelatinous  appearance.  This 
animal  comes  next  to  the  mouse  in  susceptibility,  and  cultures  which 


THE  BACILLUS   OF   ANTHRAX. 


347 


are  attenuated  to  such  an  extent  that  they  will  not  kill  a  rabbit  or  a 
sheep  may  still  kill  a  guinea-pig  ;  or,  if  not,  may  kill  a  mouse.  Pasteur 
has  shown  that  the  pathogenic  power  of  the  bacillus  may  be  reestab- 
lished by  inoculations  into  susceptible  animals,  and  that  an  attenu- 
ated culture  which  will  not  kill  an  adult  guinea-pig  may  be  fatal  to 
a  very  young  animal  of  this  species,  and  that  cultures  from  the  blood 
of  this  will  have  an  increased  pathogenic  virulence. 

Very  minute  quantities  of  a  virulent  culture  are  infallibly  fatal  to 
these  most  susceptible  animals,  but  for  rabbits  and  other  less  sus- 
ceptible animals  the  quantity  injected  influences  the  result,  and  re- 
covery may  occur  after  subcutaneous  or  intravenous  injection  of  a 
very  small  number  of  bacilli. 


FIG.  105.— Bacillus  anthracis  in  kidney  of  rabbit.    X  400.    (Baumgarten.) 

Infection  in  cattle  and  sheep  commonly  results  from  the  ingestion 
of  spores  while  grazing  in  infected  pastures.  The  bacillus  itself,  in 
the  absence  of  spores,  is  destroyed  in  the  stomach.  While  spores  are 
not  formed  in  the  bodies  of  living  animals,  their  discharges  contain 
the  bacillus,  and  this  is  able  to  multiply  in  them  and  to  form  spores 
upon  the  surface  of  the  ground  when  temperature  conditions  are 
favorable.  It  is  probable  that  this  is  the  usual  way  in  which  pastures 
become  infected,  and  that  the  bloody  discharges  from  the  bladder 
and  bowels  of  animals  suffering  from  the  disease  furnish  a  nidus  for 
the  external  development  of  these  reproductive  elements  ;  as  also  do 
the  fluids  escaping  from  the  bodies  of  dead  animals.  And  possibly, 
under  specially  favorable  conditions,  the  bacillus  may  lead  a  sapro- 
phytic  existence  for  a  considerable  time  in  the  superficial  layers  of  the 
soil. 


348  THE   BACILLUS   OF  ANTHRAX. 

Buchner  has  shown  by  experiment  that  infection  in  animals  may 
result  from  respiring  air  in  which  anthrax  spores  are  in  suspension 
in  the  form  of  dust ;  and  in  man  this  mode  of  infection  occurs  in  the 
so-called  wool-sorters'  disease. 

The  question  of  the  passage  of  the  anthrax  bacillus  from  the 
mother  to  the  foetus  in  pregnant  females  has  received  considerable 
attention.  That  this  may  occur  is  now  generally  admitted,  and  ap- 
pears to  be  established  by  the  investigations  of  Strauss  and  Chamber- 
lain, Morisani,  and  others.  That  it  does  not  always  occur  is  shown, 
however,  by  the  researches  of  other  bacteriologists,  and  especially  by 
those  of  Wolff. 

Sirena  and  Scagliosi  (1894)  report,  as  the  result  of  extended  experi- 
ments made  by  them,  that  anthrax  spores  may  survive  in  distilled 
water  for  twenty  months;  in  moist  or  dry  earth  for  two  years  and 
nine  months ;  in  sea- water  for  one  year  and  seven  months ;  in  sewage 
nearly  sixteen  months. 

Marmier  (1895)  has  made  an  extended  experimental  research  to 
determine  the  nature  of  the  specific  toxin  of  the  anthrax  bacillus. 
This  he  obtains  from  cultures,  at  a  low  temperature,  in  media  con- 
taining peptone  and  glycerin.  It  has  not  the  reactions  of  an  albu- 
minoid body  and  is  not  destroyed  by  a  temperature  of  100°  C.  In 
comparatively  large  doses  it  kills  animals  susceptible  to  anthrax,  and 
by  the  administration  of  smaller  doses  immunity  may  be  established 
in  such  animals.  This  toxin  is  contained  in  the  bacterial  cells,  and 
is  obtained  by  subjecting  these  to  the  action  of  alcohol,  or  from  the 
filtrate  when  cultures  are  made  at  a  low  temperature  in  a  medium 
containing  peptone.  It  has  not,  however,  been  obtained  in  a  pure 
form,  and  its  exact  nature  has  not  been  determined. 


VIII. 
THE  BACILLUS  OF  TYPHOID  FEVER. 

RECENT  researches  support  the  view  that  the  bacillus  described 
by  Eberth  in  1880  bears  an  etiological  relation  to  typhoid  fever — 
typhus  abdominalis  of  German  authors  ;  and  pathologists  are  dis- 
posed to  accept  this  bacillus  as  the  veritable  "germ"  of  typhoid 
fever,  notwithstanding  the  fact  that  the  final  proof  that  such  is  the 
case  is  still  wanting. 

This  final  proof  would  consist  in  the  production  in  man  or  in  one 
of  the  lower  animals  of  the  specific  morbid  phenomena  which  char- 
acterize the  disease  in  question,  by  the  introduction  of  pure  cultures 
of  the  bacillus  into  the  body  of  a  healthy  individual.  Evidently  it  is 
impracticable  to  make  the  test  upon  man,  and  thus  far  we  have  no 
satisfactory  evidence  that  any  one  of  the  lower  animals  is  subject  to 
the  disease  as  it  manifests  itself  in  man.  The  experiments  of 
Frankel  and  Simmonds  show,  however,  that  this  bacillus  is  patho- 
genic for  the  mouse  and  the  rabbit.  We  shall  refer  to  the  experi- 
ments of  these  authors  later. 

Before  the  publication  of  Eberth's  first  paper  Koch  had  observed 
this  bacillus  in  sections  made  from  the  spleen  and  liver  of  typhoid 
cases,  and  had  made  photomicrographs  from  these  sections.  His 
name  is,  therefore,  frequently  associated  with  that  of  Eberth  as  one 
of  the  discoverers  of  the  typhoid  bacillus.  Other  investigators  had  no 
doubt  previously  observed  the  same  organism,  but  some  of  them  had 
improperly  described  it  as  a  micrococcus.  Such  a  mistake  is  easily 
made  when  the  examination  is  made  with  a  low  powBr  ;  even  with  a 
moderately  high  power  the  closely  crowded  colonies  look  like  masses 
of  micrococci,  and  it  is  only  by  focussing  carefully  upon  the  scattered 
organisms  on  the  outer  margin  of  a  colony  that  the  oval  or  rod-like 
form  can  be  recognized. 

Several  observers  had  noted  the  presence  of  microorganisms  in 
the  lesions  of  typhoid  fever  prior  to  the  publication  of  Eberth 's  pa- 
per, and  Browicz  in  1875,  and  Fischel  in  1878,  had  recognized  the 
presence  of  oval  organisms  in  the  spleen  which  were  probably  identi- 
cal with  the  bacillus  of  Eberth. 

The  researches  of  Gaffky  (1884)  strongly  support  the  view  that 


350  THE   BACILLUS   OF   TYPHOID   FEVER. 

the  bacillus  under  consideration  bears  a  causal  relation  to  typhoid 
fever.  Eberth  was  only  successful  in  finding  the  bacillus  in  the 
lymphatic  glands  or  in  the  spleen  in  eighteen  cases  out  of  forty  in 
which  he  searched  for  it.  On  the  other  hand,  he  failed  to  find  it  in 
eleven  cases  of  various  nature — partly  infectious  processes — and  in 
thirteen  cases  of  tuberculosis  in  which  the  lymphatic  glands  were 
involved,  and  in  several  of  which  there  was  ulceration  of  the  mucous 
membrane  of  the  intestine. 

Koch,  independently  of  Eberth  and  before  the  publication  of  his 
first  paper,  had  found  the  same  bacillus  in  about  half  of  the  cases 
examined  by  him,  and  had  pointed  out  the  fact  that  they  were  lo- 
cated in  the  deeper  parts  of  the  intestinal  mucous  membrane,  beyond 
the  limits  of  necrotic  changes,  and  also  in  the  spleen,  whereas  the 
long,  slender  bacillus  of  Klebs  was  found  only  in  the  necrosed  por- 
tions of  the  intestinal  mucous  membrane. 

The  researches  of  W.  Meyer  (1881)  gave  a  larger  proportion  of 
successful  results.  This  author  confined  his  attention  chiefly  to  the 
swollen  plaques  of  Peyer  and  follicles  of  the  intestine  which  had  not 
yet  undergone  ulceration.  The  short  bacillus  which  had  been  de- 
scribed by  Eberth  and  Koch  was  found  in  sixteen  out  of  twenty  cases 
examined.  The  observations  of  this  author  are  in  accord  with  those 
of  Eberth  as  to  the  presence  of  the  bacillus  in  greater  abundance  in 
cases  of  typhoid  which  had  proved  fatal  at  an  early  date. 

The  fact  that  in  these  earlier  researches  the  bacilli  were  not  found 
in  a  considerable  proportion  of  the  cases  examined  is  by  no  means 
fatal  to  the  view  that  they  bear  an  etiological  relation  to  the  disease. 
As  Gaffky  says  in  his  paper  referred  to  : 

:<  This  circumstance  admits  of  two  explanations.  Either  in  those 
cases  in  which  the  bacillus  has  been  sought  with  negative  results 
they  may  have  perished  collectively,  before  the  disease  process  which 
thev  had  induced  had  run  its  course  ;  or  the  proof  of  the  presence  of 
bacilli  was  wanting  only  on  account  of  the  technical  difficulties  which 
attend  the  finding  of  isolated  colonies." 

Gaffky's  own  researches  indicate  that  the  latter  explanation  is  the 
correct  one. 

In  twenty-eight  cases  examined  by  this  author  characteristic 
colonies  of  the  bacillus  were  found  in  all  but  two.  In  one  of  these, 
one  hundred  and  forty-six  sections  from  the  spleen,  liver,  and  kid- 
neys were  examined  without  finding  a  single  colony,  and  in  the  other 
a  like  result  attended  the  examination  of  sixty-two  sections  from  the 
spleen  and  twnty-one  sections  from  the  liver.  In  the  first  of  these 
cases,  however,  numerous  colonies  were  found  in  recent  ulcers  of  the 
intestinal  mucous  membrane,  deeply  located  in  that  portion  of  the 
tissue  which  was  still  intact.  These  recent  ulcers  were  in  the  neigh- 


THE   BACILLUS   OF   TYPHOID   FEVER.  351 

borhood  of  old  ulcers  and  are  supposed  to  have  indicated  a  relapse 
of  the  specific  process.  In  the  second  case  the  negative  result  is 
thought  by  Gaffky  to  have  been  not  at  all  surprising,  as  the  patient 
died  at  the  end  of  the  fourth  week  of  sickness,  not  directly  from  the 
typhoid  process,  but  as  a  result  of  perforation  of  the  intestine. 

Gaffky  has  further  shown  that  in  those  cases  in  which  colonies 
are  not  found  in  the  spleen,  or  in  which  they  are  extremely  rare,  the 
presence  of  the  bacillus  may  be  demonstrated  by  cultivation ;  and 
that,  when  proper  precautions  are  taken,  pure  cultures  of  the  bacil- 
lus may  always  be  obtained  from  the  spleen  of  a  typhoid  case. 
Hein  has  been  able  to  demonstrate  the  presence  of  the  bacillus  and 
to  start  pure  cultures  from  material  drawn  from  the  spleen  of  a  living 
patient  by  means  of  a  hypodermatic  syringe.  Philopowicz  has  re- 
ported his  success  in  obtaining  cultures  of  the  bacillus  by  the  same 
method. 

The  fact  that  a  failure  to  demonstrate  the  presence  of  microor- 
ganisms by  a  microscopic  examination  cannot  be  taken  as  proof  of 
their  absence  from  an  organ,  is  well  illustrated  by  a  case  (No.  18)  in 
which  the  bacillus  was  obtained  by  Gaffky  from  the  spleen  and  also 
from  the  liver,  in  pure  cultures  ;  whereas  in  cover-glass  preparations 
made  from  the  same  spleen  he  failed  to  find  a  single  rod,  and  more 
than  one  hundred  sections  of  the  spleen  were  examined  before  he 
found  a  colony. 

To  obtain  pure  cultures  from  the  spleen  Gaffky  first  carefully 
washes  the  organ  with  a  solution  of  mercuric  chloride,  1 : 1,000.  A 
long  incision  is  then  made  through  the  capsule  with  a  knife  sterilized 
by  heat.  A  second  incision  is  made  in  this  with  a  second  sterilized 
knife,  and  a  third  knife  is  used  to  make  a  still  deeper  incision  in  the 
same  track.  By  this  means  the  danger  of  conveying  organisms  from 
the  surface  to  the  interior  of  the  organ  is  avoided.  From  the  bottom 
of  this  incision  a  little  of  the  soft  splenic  tissue  is  taken  up  on  a  ster- 
ilized platinum  needle,  and  this  is  plunged  into  the  solid  culture 
medium,  or  drawn  along  the  surface  of  the  same,  or  added  to  lique- 
fied gelatin  and  poured  upon  a  glass  plate.  The  colonies  develop,  in 
an  incubating  oven,  in  the  course  of  twenty-four  to  forty-eight  hours. 

Gaffky  has  also  shown  that  the  bacillus  is  present  in  the  liver,  in 
the  mesenteric  glands,  and,  in  a  certain  proportion  of  cases  at  least, 
in  the  kidneys,  in  which  it  was  found  in  three  cases  out  of  seven. 

The  appearance  of  the  colonies  in  stained  sections  of  the  spleen 
is  shown  in  Figs.  106  and  107.  Two  colonies  are  seen  in  Fig.  106 
(at  a,  a)  as  they  appear  under  a  low  power — about  sixty  diameters. 
In  Fig.  107  one  of  the  colonies  is  seen  more  highly  magnified — about 
five  hundred  diameters. 

Fraiikel  and  Simmonds  have  demonstrated  that  the  bacilli  multi- 


352 


THE  BACILLUS  OF  TYPHOID  FEVER. 


ply  in  the  spleen  after  death,  and  that  numerous  colonies  may  be 
found  in  portions  of  the  organ  which  have  been  kept  for  twenty- 
four  to  forty-eight  hours  before  they  were  placed  in  alcohol,  when 
other  pieces  from  the  same  spleen  placed  in  alcohol  soon  after  the 
death  of  the  patient  show  but  few  colonies  or  none  at  all. 

This  observation  does  not  in  any  way  weaken  the  evidence  as  to 
the  etiological  role  of  the  bacillus,  but  simply  shows  that  dead  ani- 
mal matter  is  a  suitable  nidus  for  the  typhoid  germ — a  fact  which 
has  been  repeatedly  demonstrated  by  epidemiologists  and  insisted 
upon  by  sanitarians. 

The  authors  last  referred  to  confirm  Gaff ky  as  regards  the  con- 
stant presence  of  the  bacillus  in  the  spleen.  In  twenty-nine  cases 
they  obtained  it  by  plate  cultures  twenty-five  times,  and  remark 
that  in  the  four  cases  attended  with  a  negative  result  this  result  is 


Fio.  106. 


FIG.  107. 


not  at  all  surprising,  inasmuch  as  the  typhoid  process  had  termi- 
nated and  death  resulted  from  complications. 

Gaffky  did  not  succeed  in  obtaining  cultures  from  the  blood  of 
typhoid-fever  patients,  and  concludes  from  his  researches  that  if  the 
bacilli  are  present  in  the  circulating  fluid  it  must  be  in  very  small 
numbers.  He  remarks  that  possibly  the  result  would  be  different  if 
the  blood  were  -drawn  directly  from  a  vein  instead  of  from  the  capil- 
laries of  the  skin.  Friinkel  and  Simmonds  also  report  that  gelatin, 
to  which  blood  drawn  from  the  forefinger  of  typical  cases  had  been 
added,  remained  sterile  when  poured  upon  plates  in  the  usual  man- 
ner— Koch's  method.  The  blood  was  obtained  from  six  different  in- 
dividuals,  all  in  an  early  stage  of  the  disease— the  second  to  the 
third  week.  A  similar  experiment  made  with  blood  obtained,  post 
mortem,  from  the  large  veins  or  from  the  heart,  also  gave  a  negative 
result  in  every  instance  save  one.  In  the  exceptional  case  a  single 


THE   BACILLUS    OF    TYPHOID    FEVER.  353 

colony  developed  upon  the  plate.  In  view  of  these  results  we  are 
inclined  to  attribute  the  successful  attempts  reported  by  some  of  the 
earlier  experimenters  (Letzerich,  Almquist,  Maragliano)  to  accidental 
contamination  and  imperfect  methods  of  research.  The  more  recent 
work  of  Tayon  does  not  inspire  any  greater  confidence.  This  author 
obtained  cultures  in  bouillon  by  inoculating  it  with  blood  drawn 
from  a  typhoid  patient,  and  found  that  these  were  fatal,  in  a  few 
hours,  to  guinea-pigs,  when  injected  into  the  peritoneal  cavity.  The 
lesions  observed  are  said  to  have  resembled  those  of  typhoid  fever — 
congestion  and  tumefaction  of  Peyer's  plaques  and  of  the  mesenteric 
glands,  congestion  of  the  liver,  the  kidneys,  etc. 

The  presence  of  the  bacillus  of  Eberth  in  the  alvine  evacuations  of 
typhoid  patients  has  been  demonstrated  by  Pfeiffer  and  by  Frankel 
and  Simmonds.  This  demonstration  is  evidently  not  an  easy  mat- 
ter, for  while  the  bacilli  are  probably  always  present  in  some  portion 
of  the  intestine  during  the  progress  of  the  disease,  it  does  not  follow 
that  they  are  present  in  every  portion  of  the  intestinal  contents.  As 
only  a  very  small  amount  of  material  is  used  in  making  plate  cul- 
tures, and  as  there  are  at  all  times  a  multitude  of  bacteria  of  various 
species  in  the  smallest  portion  of  faecal  matter,  it  is  not  to  be  ex- 
pected that  the  typhoid  bacillus  will  be  found  upon  every  plate. 
Frankel  and  Simmonds  made  eleven  attempts  to  obtain  the  bacillus 
by  the  plate  method,  using  three  plates  each  time,  as  is  customary 
with  those  who  adhere  strictly  to  the  directions  of  the  master,  and 
were  successful  in  obtaining  the  bacillus  in  three  instances — in  two 
in  great  numbers  and  in  the  third  in  a  very  limited  number  of  colo- 
nies. 

The  numerous  attempts  which  have  been  made  to  communicate 
typhoid  fever  to  the  lower  animals  have  given  a  negative  result  in 
every  instance.  Murchison,  in  1867,  fed  typhoid-fever  discharges  to 
swine,  and  Klein  has  made  numerous  experiments  of  the  same  kind 
upon  apes,  dogs,  cats,  guinea-pigs,  rabbits,  and  white  mice,  without 
result.  Birch-Hirschfeld,  in  1874,  by  feeding  large  quantities  of 
typhoid  stools  to  rabbits,  produced  in  some  of  them  symptoms  which 
in  some  respects  resembled  those  of  typhoid  ;  but  these  experiments 
were  repeated  by  Bahrdt  upon  ten  rabbits  with  an  entirely  negative 
result.  Von  Motschukoff sky  met  with  no  better  success  in  his  at- 
tempts to  induce  the  disease  by  injecting  blood  from  typhoid  patients 
into  apes,  rabbits,  dogs,  and  cats.  Walder  also  experimented  with 
fresh  and  with  putrid  discharges  from  typhoid  patients,  and  with 
blood  taken  from  the  body  after  death,  feeding  this  material  to 
calves,  dogs,  cats,  rabbits,  and  fowls,  without  obtaining  any  posi- 
tive results.  Klebs  has  also  made  numerous  experiments  of  a  simi- 
lar nature,  and  in  a  single  instance  found  in  a  rabbit,  which  died 
24 


354  THE   BACILLUS   OF   TYPHOID   FEVER. 

forty-seven*hours  after  receiving  a  subcutaneous  injection  of  a  cul- 
ture fluid  containing  his  "  typhoid  bacillus,"  pathological  lesions  re- 
sembling those  of  typhoid. 

Eberth  and  Gaffky  very  properly  decline  to  attach  any  import- 
ance to  this  solitary  case,  in  which,  as  the  first-named  writer  re- 
i narks,  a  different  explanation  is  possible,  and  the  possibility  of  an 
intestinal  mycosis  not  typhoid  in  its  nature  must  be  considered. 

Gaffky  has  also  made  numerous  attempts  to  induce  typhoid 
symptoms  in  animals  by  means  of  pure  cultures  of  Eberth's  bacillus, 
given  with  their  food  or  injected  into  the  peritoneal  cavity  or  subcu- 
taneously.  The  first  experiments  were  made  upon  five  Java  apes. 
For  a  considerable  time  these  animals  were  fed  daily  with  pure  cul- 
tures containing  spores.  The  temperature  of  the  animals  was  taken 
twice  daily.  The  result  was  entirely  negative.  No  better  success 
attended  the  experiments  upon  rabbits  (16),  guinea-pigs  (13),  wm'te 
rats  (7),  house  mice  (11),  field  mice  (4),  pigeons  (2),  one  hen  and  a  calf. 

Cornil  and  Babes  report  a  similar  negative  result  from  pure  cul- 
tures of  the  typhoid  bacillus  injected  into  the  peritoneal  cavity  and 
into  the  duodenum  in  rabbits  and  guinea-pigs. 

Frankel  and  Simmonds  have  made  an  extended  series  of  experi- 
ments upon  guinea-pigs,  rabbits,  and  mice,  and  have  shown  that 
pure  cultures  of  the  bacillus  of  Eberth  injected  into  the  last-men- 
tioned animals — mice  and  rabbits— may  induce  death,  and  that  the 
bacillus  may  again  be  obtained  in  pure  cultures  from  their  organs. 
It  is  not  claimed  that  the  animals  suffer  an  attack  of  typhoid  fever 
as  the  result  of  these  injections,  but  that  their  death  is  due  to  the 
i !  1 1  reduction  into  their  bodies  of  the  typhoid  bacillus,  and  that  this 
bacillus  is  thereby  proved  to  be  pathogenic. 

The  failure  to  produce  the  characteristic  lesions  of  typhoid  in  the 
lower  animals  is  evidently  not  opposed  to  the  view  that  this  bacillus 
is  the  specific  cause  of  such  lesions  in  man.  Frankel  and  Simmonds 
quote  from  Koch  in  support  of  this  statement,  as  follows  : 


in  animals  ana  in  man;  tuberculosis  does  not  present  itself  in  precisely  the 
same  manner  in  one  species  of  animals  as  in  another.  Phthisis,  as  it  occurs 
in  man.  \\c  cannot,  in  general,  produce  in  animals;  and,  nevertheless,  we 
•  •annul  assrrt  tli.it  tin-  animals  ••xp.-i-inn-iitrtl  upon  do  not  .sutler  from  tubrr- 
culoeis.  and  that  the  conclusions  which  we  draw  from  such  experiments  are 
not  perfectly  correct." 

In  Frankel  and  Simmonds'  experiments  a  considerable  quantity 
ai  material  was  used,  ,m<l  ihe  injections  were,  for  the  most  part, 
made  into  the  peritoneal  cavity  in  mice,  or  into  the  circulation 


THE   BACILLUS    OF   TYPHOID    FEVER.  355 

through  a  vein  in  rabbits.  The  influence  of  quantity  of  material 
used  is  especially  shown  in  the  case  of  the  mice,  and  the  question 
arises  whether  the  pathogenic  power  of  the  bacillus  for  these  animals 
does  not  depend  upon  the  simultaneous  injection  of  the  ptomaine  de- 
veloped 4n  cultures  as  a  result  of  the  vital  activity  of  the  organism. 
Thus  we  read  that  mouse  No.  4  resisted  an  injection  of  a  diluted  cul- 
ture, No.  1,  but  succumbed  to  a  more  concentrated  suspension — one- 
fifth  of  a  Pravaz  syringe.  Mouse  No.  5  was  not  killed  by  the 
injection  of  one-third  of  a  syringeful  of  a  diluted  culture,  but  subse- 
quently died  from  the  injection  of  one-third  of  a  syringeful  of  an 
undiluted  culture.  Mouse  No.  16,  injected  October  10th  with  half 
of  a  syringeful  of  a  very  diluted  culture,  did  not  die.  The  injection 
was  repeated  on  the  17th  of  October  with  half  a  syringeful  of  an  un- 
diluted culture,  with  fatal  result. 

In  all,  thirty-five  mice  were  injected,  with  a  fatal  result  in  twen- 
ty-seven cases.  In  rabbits  the  injections  were '  commonly  made  in 
the  large  vein  of  the  ear,  and  the  quantity  of  material  injected  was 
considerably  greater — from  one-third  the  contents  of  a  hypodermatic 
syringe  to  two  syringefuls.  In  some  instances  death  occurred  with- 
in a  few  hours,  in  others  on  the  following  day  or  after  an  interval  of 
two  or  three  days.  It  is  noticeable  that  the  results  differ  very  great- 
ly as  to  the  date  of  death  and  the  relative  quantity  of  material  re- 
quired to  produce  a  fatal  result.  This  probably  depends  to  some 
extent  upon  the  size  of  the  animal,  and  perhaps  partly  upon  indi- 
vidual differences  in  resisting  power. 

The  experiments,  considered  together,  show  that  the  typhoid  ba- 
cillus is  not  pathogenic  for  these  animals  in  the  same  sense  as  is  the 
anthrax  bacillus  or  the  bacillus  of  rabbit  septica3mia.  These  organ- 
isms introduced  beneath  the  skin  or  into  the  circulation  in  the  small- 
est amount  infallibly  produce  death,  and  at  the  expiration  of  a  pe- 
riod of  time  which  is  tolerably  uniform. 

In  all,  seventy-nine  experiments  upon  rabbits  were  made,  with  the 
following  result  :  Five  injections  into  the  intestine,  five  into  the  sub- 
cutaneous connective  tissue,  one  into  the  lung,  and  two  inhalation 
experiments,  all  without  result;  twenty  injections  into  the  peri- 
toneal cavity  furnished  two,  and  forty-six  injections  into  the  vein  of 
the  ear  twenty  positive  results — i.  e. ,  were  fatal  to  the  animal. 

In  the  fatal  cases  the  bacilli  were  proved  to  be  present  in  the 
spleen  by  culture  experiments  and  by  microscopical  examination  of 
properly  stained  sections.  The  colonies  were  identical  in  appearance 
with  those  found  in  the  spleen  of  cases  of  typhoid  in  man.  Col- 
onies were  found  in  the  spleens  of  the  rabbits  experimented  upon 
exactly  as  in  the  human  subject — sometimes  in  the  trabecuhe,  some- 
times in  the  Malpighian  bodies,  sometimes  free  in  the  splenic  pulp. 


356  THE   BACILLUS   OF   TYPHOID   FEVER. 

Brieger  has  made  some  very  interesting  researches  with  reference 
to  the  chemical  substances  which  are  produced  as  a  result  of  the 
physiological  processes  attending  the  growth  of  this  bacillus. 

Having  obtained  a  culture  from  the  spleen  of  a  typhoid  patient, 
and  assured  himself  by  comparison  with  a  pure  culture  given  him 
by  Koch  that  he  was  dealing  with  the  right  organism,  Brieger 
planted  the  bacillus  in  a  culture  solution  containing  grape  sugar  and 
salts— Nahrsalzen— in  which  it  thrived  admirably.  Such  a  solution 
at  30°  C.  became  clouded  at  the  end  of  twenty-four  hours,  and  gave 
off  an  evident  odor  of  ethyl  alcohol,  which  increased  from  day  to  day. 
In  addition  to  ethyl  alcohol  small  quantities  of  the  volatile  fat  acids 
were  produced— among  them  acetic  acid.  Lactic  acid  was  also 
formed  from  the  grape  sugar.  The  bacillus  grew  still  better  in  al- 
buminous culture  fluids.  It  did  not  in  these  give  rise  to  the  produc- 
tion of  sulphuretted  hydrogen  or  of  any  of  the  volatile  products  of 
putrefactive  decomposition,  such  as'  indol  and  phenol.  There  was 
no  gas  formation  in  such  cultures,  even  after  standing  for  eight 
weeks,  but  a  slight  odor,  resembling  that  of  whey,  was  given  off 
from  the  cultures.  Repeatedly,  but  not  in  every  case,  Brieger  suc- 
ceeded in  obtaining  from  such  cultures  a  very  deliquescent  basic 
product.  This  was  obtained  in  only  very  small  quantities,  even 
when  the  cultures  had  remained  in  the  incubating  oven  for  a  month. 
The  physiological  properties  of  this  base  have  convinced  Brieger  that 
it  is  a  new  ptomaine.  In  guinea-pigs  this  ptomaine  produced  a  slight 
flow  of  saliva  and  frequent  respiration.  Later  the  animals  lost  con- 
trol of  their  extremities  and  of  the  muscles  of  the  trunk  ;  they  fell 
upon  their  side,  but  when  placed  upon  their  legs  were  able  to  move 
forward  a  little  ;  they,  however,  soon  fell  again  and  remained  help- 
less upon  their  side.  The  pupils  gradually  became  widely  dilated 
and  failed  to  respond  to  light ;  the  flow  of  saliva  became  more  pro- 
fuse ;  no  convulsions  occurred.  Little  by  little  the  pulsations  of  the 
heart  and  the  breathing  became  more  frequent.  During  the  entire 
course  of  these  symptoms  the  animals  had  frequent  liquid  discharges. 
Death  occurred  in  from  twenty-four  to  forty-eight  hours.  Post- 
mortem examination  showed  the  heart  to  be  contracted  in  systole, 
the  lungs  to  be  hypersemic,  the  intestine  contracted  and  pale. 

The  experimental  evidence  which  we  have  presented,  considered 
in  connection  with  established  facts  relating  to  the  propagation  of 
typhoid  fever,  seems  to  the  writer  to  be  convincing  as  regards  the 
etiological  role  of  this  bacillus. 

No  other  organism  has  been  found,  after  the  most  careful  search, 
in  the  deeper  portions  of  the  intestinal  glands  involved  in  this  disease, 
"i-  in  the  internal  organs  ;  on  the  other  hand,  this  bacillus  has  been 
demonstrated  to  be  constantly  present.  It  is  undoubtedly  present 
Cr  •  f^n 


THE   BACILLUS    OF   TYPHOID    FEVER.  357 

during  the  lifetime  of  the  patients,  and  is  found  in  greater  abun- 
dance in  those  cases  which  terminate  fatally  at  an  early  date.  It  is 
not  a  putrefactive  organism,  and  is  not  developed  in  the  tissues  post 
mortem,  although  it  has  been  shown  by  Frankel  and  Simmonds  that 
it  multiplies  rapidly  in  the  spleen  after  death,  up  to  the  time  that 
putrefactive  decomposition  commences.  This  is  quite  in  accord  with 
what  we  should  a  priori  have  expected,  in  view  of  the  facts  relat- 
ing to  the  propagation  of  typhoid  fever.  These  facts  indicate  that 
the  disease  in  question  is  due  to  a  microorganism  which  is  capable  of 
multiplication  external  to  the  human  body  in  a  variety  of  organic 
media,  at  comparatively  low  temperatures,  and  that  it  is  widely  dis- 
tributed. From  the  endemic  prevalence  of  the  disease  over  vast 
areas  of  the  earth's  surface  we  may  infer  that  it  is  induced  by  a 
hardy  microorganism.  Eberth's  bacillus  complies  with  all  of  these 
conditions. 

There  are  numerous  facts  which  indicate  that  the  development  of 
an  attack  of  typhoid  and  the  severity  of  the  symptoms  depend  to 
some  extent  upon  the  quantity  of  the  infectious  material  introduced 
into  the  alimentary  canal.  Milk  or  water  which  has  been  infected 
directly  by  the  discharges  of  typhoid  patients  is  especially  danger- 
ous, and  there  is  reason  to  believe  that  repeated  or  concentrated 
doses  of  such  infectious  material  may  be  effective  when  a  single 
draught  of  the  contaminated  fluid,  or  a  greater  degree  of  dilution, 
would  be  innocuous. 

Again,  we  have  evidence  that  the  typhoid  germs  may  become 
effective  as  a  result  of  certain  favorable  circumstances  relating  to  the 
individual  or  to  his  environment.  Those  agencies  which  reduce  the 
vital  resisting  power  of  the  tissues,  and  especially  exposure  to  the 
emanations  from  putrefying  material,  to  sewer  gas,  to  vitiated  air  in 
overcrowded  and  ill- ventilated  apartments,  etc.,  are  recognized  as 
favorable  to  the  development  of  typhoid  fever  where  the  specific 
cause  is  present.  All  these  facts  seem  to  accord  with  the  experi- 
mental evidence  which  indicates  that  the  pathogenic  power  of  the 
bacillus  of  Eberth  depends  upon  the  formation  of  a  poisonous 
ptomaine  rather  than  upon  a  special  facility  for  multiplying  in  the 
tissues  of  a  living  animal.  Indeed,  it  seems  quite  probable  that  its 
power  to  invade  living  animal  tissues  depends  upon  the  toxic  action 
of  this  ptomaine  ;  or,  it  may  be,  of  other  ptomaines  produced  under 
certain  circumstances  in  the  body  in  excess  or  introduced  from  with- 
out. Such  toxic  agents  may  serve,  when  the  specific  germ  is  intro- 
duced into  the  intestine  in  comparatively  small  numbers,  to  give  it 
the  mastery  over  the  vital  resisting  power  of  the  tissues  subject  to  in- 
vasion, and  thus  to  induce  an  attack  of  the  disease. ' 

1  The  above  account  of  researches  relating  to  the  etiology  of  typhoid  fever  is  from 


358 


THE   BACILLUS   OF   TYPHOID    FEVER. 


Hi.     BACILLUS   TYPHI   ABDOMINALIS. 

Synonyms. — Bacillus  typhosus  ;  Typhus  bacillus. 

Eberth  (1880  and  1881)  demonstrated  the  presence  of  this  bacillus 
in  the  spleen  and  diseased  glands  of  the  intestine  in  typhoid  cada- 
vers. Gaffky  (1884)  first  obtained  it  in  pure  cultures  from  the  same 
source  and  determined  its  principal  biological  characters. 

It  is  found,  in  the  form  of  small,  scattered  colonies,  in  the  spleen, 
the  liver,  the  glands  of  the  mesentery,  the  diseased  intestinal  glands, 
and  in  smaller  numbers  in  the  kidneys,  in  fatal  cases  of  typhoid  fever; 
it  has  also  been  obtained,  by  puncture,  from  the  spleen  during  life, 
from  the  alvine  discharges  of  the  sick,  and  rarely  from  the  urine. 
It  is  not  found  in  the  blood  of  the  general  circulation,  unless,  pos- 
sibly, in  rare  cases  and  in  small  numbers. 


Fio.  108.  FIG.  109. 

Fio.  108.— Bacillus  typhi  abdominalis,  from  single  gelatin  colony,  x  1,000.  From  a  photo- 
micrograph.  (Frfinkel  and  Pfeiffer. ) 

Fio.  109  —Bacillus  typhi  abdominalis,  from  single  gelatin  colony.  X  1,000.  From  a  photo- 
micrograph. (Sternberg.) 

Morphology. — Bacilli,  usually  one  to  three  /*  in  length  and  about 
0.5  to  0.8  /*  broad,  with  rounded  ends  ;  may  also  grow  out  into  long 
threads,  especially  upon  the  surface  of  cooked  potato.  The  dimen- 
sions of  the  rods  differ  considerably  in  different  media.  Spherical  or 
oval  refractive  granules  are  often  seen  at  the  extremities  of  the  rods, 
•  •>|MM-ially  in  potato  cultures  kept  in  the  incubating  oven;  these  are 
not  reproductive'  spores,  as  was  at  first  supposed.  The  bacilli  have 
numerous  flagella  arranged  around  the  periphery  of  the  cells — usually 
from  five  to  twenty,  but  many  short  rods  have  but  a  single 

a  paper  read  by  t lie  writer  :it  the  annual   meeting  of  the  Association  of  American 
Physicians,  Washington,  D.  C.,  June  18th,  1886. 


THE   BACILLUS   OF   TYPHOID    FEVER. 


359 


terminal  flagellum.  These  flagella  are  spiral  in  form,  about  0. 1  p.  in 
thickness,  and  from  three  to  five  times  as  long  as  the  rods  (Babes). 

In  stained  preparations  unstained  "  vacuoles"  may  often  be  seen 
at  the  margins  of  the  rods,  either  along  the  sides  or  at  the  ends  ; 
these  appear  to  be  due  to  a  retraction  of  the  protoplasm  from  the  cell 
membrane. 

The  typhoid  bacillus  stains  with  the  aniline  colors,  but  more 
slowly  than  many  other  bacteria,  and  easily  parts  with  its  color  when 
treated  with  decolorizing  agents — e.g.,  iodine  solution  as  employed  in 
Gram's  method.  Loffler's  solution  of  methylene  blue  is  an  excellent 
staining  agent  for  this  bacillus,  but  permanent  preparations  fade  out 
after  a  time  ;  f uchsin,  gentian  violet,  or  Bismarck  brown,  in  aqueous 
solution,  may  also  be  used.  The  flagella  may  be  demonstrated  by 
Lomer's  method  of  staining  (p.  32). 


3£*      \       -Sx.'^ 


FIG.  110.— Bacillus  typhi  abdominalis.  stained  by  Loffler's  method,  showing  flagella.    x  1,000. 
From  a  photomicrograph  by  Frankel  and  Pfeiffer. 

To  stain  the  bacillus  in  sections  of  the  spleen,  etc.,,  it  is  best  to 
leave  these  in  Loffler's  methylene  blue  solution  or  in  the  carbol- 
fuchsin  solution  of  Ziehl  for  twelve  hours  or  more ;  or  the  aniline- 
fuchsin  solution  may  be  used.  The  sections  should  be  washed  in 
distilled  water  only,  when  ZiehFs  solution  is  used,  or  with  a  very  di- 
lute solution  of  acetic  acid  when  Ehrlich's  tubercle  stain  is  employed 
(Baumgarten). 

Biological  Characters. — The  typhoid  bacillus  is  a  motile,  aero- 
bic, non-liquefying  bacillus,  which  grows  readily  in  a  variety  of 
culture  media  at  the  "  room  temperature."  Although  it  grows  most 
abundantly  in  the  presence  of  free  oxygen,  it  may  also  develop  in  its 
absence,  and  is  consequently  a  facultative  anaerobic. 


360 


THE  BACILLUS   OF   TYPHOID   FEVER. 


FIG.  111.— Single  colony  of  Bacillus 
typhi  abdominalis,  in  nutrient  gela- 
tin, (x?)  From  a  photograph  by 
ROUT. 


In  gelatin  plate  cultures  small,  white  colonies  are  developed  at 

the  end  of  thirty-six  to  forty-eight  hours,  which  under  the  microscope 

are  seen  to  be  somewhat  irregular  in 
outline  and  of  a  spherical,  oval,  or  long- 
oval  form  ;  these  have  by  transmitted 
light  a  slightly  granular  appearance  and 
a  yellowish-brown  color.  At  the  end  of 
three  or  four  days  the  colonies  upon  the 
surface  of  the  gelatin  form  a  grayish- 
white  layer  of  one  to  two  millimetres  in 
diameter,  with  more  or  less  irregular 
margins,  and,  when  developed  from  deep 
colonies,  with  an  opaque  central  nucleus. 
These  colonies,  by  transmitted  light, 
have  a  yellowish-brown  color  towards 
the  centre,  where  they  are  thickest, 

while  the  margins  are  colorless  and  transparent  ;  the  surface  is  com- 

monly marked  with  a  network  of  lines  and  furrows.     Stick  cultures 

in  ten-per-cent  gelatin,  at  18°  to  20°  C.,  at  the 

end  of  three  days  show  upon  the  surface  a 

whitish,  semi-transparent  layer,  with  sharply 

defined  margins  and  irregular  outline,  which 

has  a  shining,  pearly  lustre  ;  and  along  the 

line  of  puncture  a  grayish-  white  growth,  made 

up  of  crowded  colonies,  which  are  larger  and 

more  distinct  at  the  bottom  of  the  line  of  growth. 

Upon  nutrient  agar,  at  a  temperature  of  35° 

to  37°  C.,  the  growth  is  more  rapid  and  forms 

a  whitish,  semi-transparent  layer.     The  cul- 

tures give  off  a  faint  putrefactive  odor.    The 

growth  upon  blood  serum  is  rather  scanty,  in 

the  form  of  transparent,  shining  patches  along 

the  line  of  inoculation. 

The  typhoid  bacillus  develops  abundantly 

in  milky  in  which  fluid  it  produces  an  acid  re- 

action ;  it  also  grows  in  various  vegetable  in- 

fusions and  in  bouillon. 

None  of  the  above  characters  of  growth 

are  distinctive,  as  certain  common  bacilli  found 

in  normal  faeces  present  a  very  similar  appear- 

anre  U'hrri  cultivated  in  tho  same  media. 

The  growth  of  this  bacillus  upon  potato  is 
an  important  character,  as  was  first  pointed  out 
by  Gaffky,  In  the  incubating  oven  at  the  end  of  forty-eight  hours, 


FIG   m._Bacillua  typhi 

abdominalis;   stick  culture 


THE   BACILLUS    OF   TYPHOID    FEVER.  361 

or  at  the  room  temperature  in  three  or  four  days,  the  surface  of 
the  potato  has  a  moist,  shining  appearance,  but  there  is  no  visible 
growth  such  as  is  produced  by  many  other  bacteria  upon  this  me- 
dium. A  simple  inspection  would  lead  to  the  belief  that  no  growth 
had  occurred;  but  if  with  a  platinum  needle  a  little  material  is 
scraped  from  any  portion  of  the  shining  surface  and  a  stained  pre- 
paration is  made  from  it,  numerous  bacilli  will  be  seen,  some  of 
which  are  likely  to  be  in  the  form  of  quite  long  threads,  while  others 
are  short  and  have  rounded  extremities.  This  "invisible  growth" 
has  been  shown  by  the  researches  of  Buchner  and  others  to  be  most 
characteristic  upon  potatoes  having  a  decidedly  acid  reaction,  as  is 
usually  the  case.  When  cultivated  upon  potatoes  having  an  alkaline 
reaction  a  thin,  visible  film  of  a  yellowish-brown  color  and  of  limited 
extent  may  be  developed.  Inasmuch  as  several  common  and  widely 
distributed  bacteria  closely  resemble  the  typhoid  bacillus  in  form  and 
in  their  growth  in  nutrient  gelatin,  this  character  of  invisible  growth 
upon  potato  is  very  important  for  its  differentiation,  especially  as  the 
common  bacilli  referred  to — Bacillus  coli  communis,  bacillus  of  Em- 
merich— produce  a  very  distinct  and  rather  thick,  yellowish- white 
mass  upon  the  surface  of  potato.  But  recent  researches  show  that 
this  invisible  growth,  although  not  a  common  character,  does  not 
belong  exclusively  to  the  typhoid  bacillus  (Babes). 

This  bacillus  in  its  development  in  culture  media  produces  acids — 
according  to  Brieger  small  quantities  of  volatile  fat  acids,  and,  in 
presence  of  grape  sugar,  lactic  acid.  It  also  grows  readily  in  a  de- 
cidedly acid  medium,  and  this  character  has  been  employed  as  a  test 
for  differentiating  it  from  other  similar  bacilli ;  but  some  of  these 
also  grow  in  a  decidedly  acid  medium,  and  too  much  reliance  cannot 
be  placed  upon  this  test. 

Brieger  has  shown  that  indol  is  not  produced  in  cultures  of  the 
typhoid  bacillus,  and  Kitasato  has  proposed  to  use  the  indol  test  for 
differentiating  this  from  other  similar  bacilli  which  are  said,  as  a 
rule,  to  give  the  indol  reaction.  This  test  consists  in  the  addition  to 
ten  cubic  centimetres  of  a  bouillon  culture  which  has  been  in  the  in- 
cubating oven  for  twenty-four  hours,  of  one  cubic  centimetre  of  a 
solution  of  sodium  nitrite  (0. 02  gramme  to  one  hundred  cubic  centi- 
metres of  distilled  water),  together  with  a  few  drops  of  concentrated 
sulphuric  acid.  If  indol  is  present  a  red  color  is  developed. 

None  of  the  above-mentioned  tests  are  entirely  reliable,  but,  taken 
together  with  the  morphological  and  biological  characters  above  de- 
scribed, they  may  enable  the  bacteriological  expert  to  give  a  tolerably 
confident  opinion  as  to  the  presence  of  this  bacillus  in  a  water  supply 
suspected  of  contamination,  etc.  And  when  a  bacillus  having  these 
characters  is  obtained  in  a  pure  culture  from  the  spleen  of  a  typhoid 


THE  BACILLUS   OF   TYPHOID   FEVER. 

cadaver  the  student  may  be  very  sure  that  he  has  the  typhoid  bacillus. 
But  in  the  presence  of  various  similar  bacilli,  as  in  faeces,  very  careful 
comparative  researches  will  be  required  to  determine  in  a  definite 
manner  that  a  non-liquefying  bacillus  obtained  in  pure  cultures  by 
the  plate  method  is  really  the  one  now  under  consideration— espe- 
cially so  as  the  cultures  of  the  typhoid  bacillus  in  the  same  medium 
may  differ  considerably  at  different  times,  and  a  number  of  bacilli 
are  known  which  resemble  it  so  closely  that  it  is  still  uncertain 
whether  they  are  to  be  considered  as  varieties  of  the  typhoid  bacillus 
or  as  distinct  species.  Thus  Babes,  in  an  extended  research,  found  in 
the  organs  of  typhoid  cases,  associated  with  the  true  typhoid  bacillus, 
other  bacilli  or  varieties  very  closely  resembling  it.  He  has  also 
described  three  varieties  (?),  obtained  by  him  from  other  sources, 
which  could  only  be  differentiated  from  the  true  typhoid  bacillus  by 
very  careful  comparison  of  cultures  made  side  by  side  in  various 
media. 

Cassedebat,  also,  in  an  extended  examination  of  the  river  water 
at  Marseilles  with  reference  to  the  presence  of  the  typhoid  bacillus, 
found  three  species  which  very  closely  resembled  it,  but  which  by 
careful  comparison  were  shown  to  present  slight  but  constant  dif- 
ferences in  their  biological  characters.  He  was  not  able  to  find  the 
true  typhoid  bacillus,  and  his  researches,  together  with  those  of  Babes 
and  other  recent  investigators,  make  it  appear  probable  that  numerous 
mistakes  have  been  made  by  bacteriologists  who  have  reported  the 
finding  of  the  typhoid  bacillus  in  river  and  well  water,  in  faeces,  etc., 
and  who  have  depended  mainly  upon  the  character  of  invisible 
growth  upon  potato  in  making  their  diagnosis.  Cassedebat  states 
that  all  three  of  his  pseudo-typhoid  bacilli  corresponded  in  their 
growth  upon  potato  with  the  bacillus  of  Eberth.  They  also  corre- 
sponded in  their  growth  on  gelatin,  agar-agar,  and  blood  serum, 
which,  as  heretofore  remarked,  has  no  characteristic  features.  They 
all  gave  a  negative  indol  reaction.  Like  the  typhoid  bacillus,  they 
grew  in  milk  without  causing  coagulation  of  the  casein,  but  two  of 
them  produced  an  alkaline  reaction  in  this  fluid,  while  the  third  cor- 
responded with  the  typhoid  bacillus  in  producing  a  decided  acid  re- 
action. Differences  were  also  observed  in  bouillon  cultures,  and  in 
bouillon  and  milk  to  which  various  aniline  colors  had  been  added,  as 
recommended  by  Holz. 

Whether  the  typhoid  bacillus,  as  obtained  from  the  spleen  of  a 
typhoid  cadaver,  is  in  truth  specifically  distinct  from  these  similar 
bacilli,  or  whether  they  are  all  varieties  of  the  same  species,  result- 
ing from  modifications  in  their  biological  characters  acquired  during 
their  continuous  development  under  different  conditions,  is  an  un- 
si-ttl.-.l  qu.-Miuii.  Jiut,  in  view  of  the  experimental  evidence  now 


THE   BACILLUS   OF   TYPHOID   FEVER.  363 

available,  there  is  nothing  improbable  in  the  supposition  that  they  are 
simply  varieties,  and  that,  as  the  result  of  a  saprophytic  mode  of 
life,  this  bacillus  may  undergo  more  or  less  permanent  modifications. 

In  the  writer's  experiments  (1887)  the  thermal  death-point  of  the 
typhoid  bacillus  was  found  to  be  56°  C.,  the  time  of  exposure  being 
ten  minutes  ;  and  potato  cultures  containing  the  refractive  granules 
described  by  Gaffky  as  spores  were  found  to  be  infallibly  destroyed 
by  a  temperature  of  60°  C.  This  result  has  been  confirmed  by  Buch- 
ner  (1888)  and  by  Janowsky  (1890),  and  the  inference  seems  justified 
that  these  granules  are  not  reproductive  bodies,  as  was  at  first  be- 
lieved ;  for  spores  are  distinguished  by  their  great  resistance  to  heat 
and  other  destructive  agencies.  According  to  Buchner,  the  bacilli 
containing  these  refractive  granules  are  even  less  resistant  than  fresh 
cultures  in  which  they  are  not  present,  and  he  is  disposed  to  look 
upon  them  as  representing  a  degeneration  of  the  protoplasm  of  the 
cells.  They  do  not  stain  by  the  methods  which  are  successful  in 
staining  the  spores  of  other  bacilli,  and,  in  short,  present  none  of  the 
characters  which  distinguish  spores,  except  the  form  and  high  re- 
fractive power. 

The  typhoid  bacillus  retains  its  vitality  for  many  months  in  cul- 
tures; the  writer  has  preserved  bouillon  cultures  for  more  than  a  year 
in  hermetically  sealed  tubes,  and  has  found  that  development 
promptly  occurred  in  nutrient  gelatin  inoculated  from  these.  Dried 
upon  a  cover  glass,  it  may  grow  in  a  suitable  medium  after  having 
been  preserved  for  eight  to  ten  weeks  (Pfuhl).  When  added  to 
sterilized  distilled  water  it  may  retain  its  vitality  for  more  than  four 
weeks  (Bolton),  (forty  days  Cassedebat),  and  in  sterilized  sea- water 
for  ten  days  (De  Giaxa).  Added  to  putrefying  faeces  it  may  preserve 
its  vitality  for  several  months  (Ufflemann),  in  typhoid  stools  for  three 
months  (Karlinski),  and  in  earth  upon  which  bouillon  cultures  had 
been  poured  for  five  and  one-half  months  (Grancher  and  Deschamps). 

In  hanging-drop  cultures  this  bacillus  may  be  seen  to  exhibit  very 
active  movements,  the  shorter  rods  rapidly  crossing  the  field  with  a 
darting  or  to-and-fro,  progressive  motion,  while  longer  filaments 
move  in  a  serpentine  manner. 

In  addition  to  the  volatile  fat  acids  which,  according  to  Brieger, 
are  formed  in  small  amounts  in  cultures  of  the  typhoid  bacillus,  and 
to  lactic  acid  formed  in  solutions  containing  grape  sugar,  a  basic 
substance  possessing  toxic  properties  has  been  isolated  by  the  chemist 
named — his  typhotoxine  (C,Hl7N"Oa).  Brieger  supposes  that  other 
basic  substances  are  likewise  formed,  but  believes  this  to  be  the  speci- 
fic product  to  which  the  pathogenic  action  of  the  bacillus  is  due.  It 
is  a  strongly  alkaline  base,  which  produces  in  mice  and  guinea-pigs 
salivation,  paralysis,  dilated  pupils,  diarrhoea,  and  death. 


364  THE  BACILLUS  OF  TYPHOID  FEVER. 

Numerous  experiments  have  been  made  to  determine  the  amounts 
of  various  germicidal  agents  required  to  destroy  the  vitality  of  this 
bacillus,  and  the  action  of  antiseptics  in  restraining  its  development. 
For  the  results  of  these  experiments  the  reader  is  referred  to  the 
sections  in  Part  Second  relating  to  the  action  of  antiseptics  and  disin- 
fectants. 

Pathogenesis.—  The  very  numerous  experiments  which  have  been 
made  on  the  lower  animals  have  not  been  successful  in  producing  in 
any  one  of  them  a  typical  typhoid  process.  Nor  is  this  surprising, 
in  view  of  the  fact  that,  so  far  as  is  known,  no  one  of  them  is  liable  to 
contract  the  disease,  as  man  does,  by  the  use  of  infected  food  or 
water. 

The  experiments  of  Frankel  and  Simmonds  show  that  when  con- 
siderable quantities  of  a  pure  culture  of  this  bacillus  are  injected  into 


Fia    118— Section  through  wall  of  intestine,  showing  invasion  by  typhoid  bacilli.    X  950. 
(Baumgarten.) 

the  circulation  of  rabbits  through  the  ear  vein,  or  into  the  peritoneal 
cavity  of  mice,  a  certain  proportion  of  the'  inoculated  animals  die, 
usually  within  forty-eight  hours,  and  that  the  bacillus  may  be  re- 
covered from  the  various  organs,  although  it  is  not  present  in  the 
blood.  But  death  does  not  always  occur  from  intravenous  injections, 
and  subcutaneous  or  intraperitoneal  injections  in  rabbits  are  usually 
without  result.  Subcutaneous  injections  in  mice  proved  to  be  fatal  in 
ten  cases  out  of  sixteen  inoculated  by  A.  Frankel.  Seitz,  by  following 
Koch's  method — i.e.,  by  rendering  the  contents  of  the  stomach  alka- 
line, and  arresting  intestinal  peristalsis  by  the  administration  of 
opium — obtained  a  fatal  result,  in  a  majority  of  the  guinea-pigs  experi- 
mented upon,  from  the  introduction  of  ten  cubic  centimetres  of  a 
bouillon  culture  into  the  stomach  through  a  pharyngeal  catheter. 
We  may  remark,  with  reference  to  these  results,  that  while  they  show 
that  cultures  of  the  typhoid  bacillus  have  a  certain  pathogenic  power, 


THE   BACILLUS   OF   TYPHOID   FEVER.  365 

they  also  show  that  the  animals  experimented  upon  frequently  re- 
covered after  comparatively  large  doses,  and  that  the  typhoid  bacil- 
lus is  not  pathogenic  in  the  same  sense  as  are  those  microorganisms 
which,  when  introduced  into  the  body  of  a  susceptible  animal  in  very 
minute  amount,  give  rise  to  general  infection  and  death.  On  the 
other  hand,  a  fatal  result  depends  upon  the  quantity  of  the  culture 
introduced  in  the  first  instance,  rather  than  upon  the  multiplication 
of  the  bacillus  in  the  body  of  the  inoculated  animal.  This  view  is 
confirmed  by  the  experiments  of  Sirotinin,  which  show  not  only  that 
a  fatal  result  depends  upon  the  quantity  injected,  but  also  that  a 
similar  result  follows  the  injection  of  cultures  which  have  been  ster- 
ilized by  heat  or  filtration.  The  pathogenic  action,  then,  depends 
upon  the  presence  of  toxic  substances  produced  during  he  growth  of 
the  bacillus  in  artificial  culture  media.  The  researches  of  Brieger, 
heretofore  referred  to,  show  the  presence  in  such  cultures  of  a  toxic 
ptomaine — his  typhotoxine — to  which  the  pathogenic  potency  of  these 
cultures  appears  to  be  due.  White  mice  and  guinea-pigs  usually  die 
in  from  twenty-four  to  forty-eight  hours  when  inoculated  in  the 
cavity  of  the  abdomen  with  a  virulent  culture  of  the  typhoid  bacillus 
— 0. 1  cubic  centimetre  to  0. 5  cubic  centimetre  of  a  bouillon  culture 
three  days  old.  According  to  Kitasato,  the  virulence  of  cultures 
from  different  cases  of  typhoid  fever  varies  considerably. 

Detection  of  the  Typhoid  Bacillus  in  Water. — The  generally 
recognized  fact  that  typhoid  fever  is  usually  contracted  by  drink- 
ing water  contaminated  by  the  typhoid  bacillus  has  led  to  numer- 
ous researches  having  for  their  object  the  discovery  of  a  reliable 
method  of  detecting  this  bacillus  when  present  in  water  in  compara- 
tively small  numbers  in  association  with  the  ordinary  water  bacilli. 
The  use  of  Koch's  plate  method,  as  commonly  employed,  will 
not  suffice,  because  the  water  bacilli  present  grow  more  rapidly 
and  cause  liquefaction  of  the  gelatin  before  visible  colonies  of  the 
typhoid  bacillus  are  formed  ;  and,  owing  to  the  relatively  small 
number  of  typhoid  bacilli,  these  are  likely  to  escape  detection.  The 
aim  of  bacteriologists  has,  therefore,  been  to  restrain  the  growth  of 
these  common  water  bacilli  by  some  agent  which  does  not  at  the 
same  time  prevent  the  development  of  the  typhoid  bacillus.  Chan- 
temesse  and  Widal  were  the  first  to  propose  the  use  of  carbolic  acid 
for  this  purpose.  They  recommended  the  addition  of  0. 25  per  cent 
of  this  agent  to  nutrient  gelatin  ;  but,  according  to  Kitasato,  the  de- 
velopment of  the  typhoid  bacillus  is  restrained  by  an  amount  exceed- 
ing 0. 20  per  cent. 

Holz  prepares  an  acid  medium  by  adding  gelatin  (ten  per  cent)  to 
the  juice  of  raw  potatoes,  and  asserts  that  while  the  typhoid  bacillus 
grows  luxuriantly  in  this  medium,  many  other  bacilli  fail  to  develop 


;},;,;  THE  BACILLUS   OF   TYPHOID    FEVER. 

in  it.  The  test  is  said  to  be  still  more  reliable  if  0.05  per  cent  of  car- 
bolic acid  is  added  to  the  "  potato-gelatin."  According  to  Holz,  the 
addition  of  more  than  0.1  percent  of  carbolic  acid  to  nutrient  gelatin 
prevents  the  free  development  of  the  typhoid  bacillus. 

Thoinothas  claimed  to  be  able  to  obtain  the  typhoid  bacillus  from 
mixed  cultures— as,  for  example,  from  faeces— by  suspending  a  small 
amount  of  material  containing  it  for  several  hours  in  a  solution  con- 
taining 0.25  per  cent  of  carbolic  acid.  While  other  bacilli  are 
destroyed,  the  typhoid  bacillus  is  said  to  survive  such  exposure. 

The  method  of  Parietti  has  recently  been  tested  in  a  practical 
way  by  Kamen,  and  proved  to  be  satisfactory  for  the  detection  of 
the  typhoid  bacillus  in  water  which  was  supposed  to  be  the  source  of 
a  local  epidemic  of  the  disease.  The  following  solution  is  used  : 

Carbolic  acid, 5  grammes. 

Hydrochloric  acid  (pure), 4 

Distilled  water, 100 

Several  test  tubes,  each  of  which  contains  ten  cubic  centimetres 
of  neutral,  sterilized  bouillon,  are  used  in  the  experiment.  From 
three  to  nine  drops  of  the  acid  solution  are  added  to  each  of  these, 
and  the  tubes  are  then  placed  in  an  incubating  oven  for  twenty-four 
hours  to  ascertain  whether  they  are  still  sterile  after  this  addition. 
If  the  bouillon  remains  clear,  from  one  to  ten  drops  of  the  suspected 
water  are  added  to  each  tube  and  they  are  returned  to  the  incubating 
oven.  If  at  the  end  of  twenty-four  hours  the  bouillon  becomes 
clouded,  this  is  due,  according  to  Parietti,  to  the  presence  of  the 
typhoid  bacillus,  which  is  then  to  be  obtained  in  pure  cultures  by  the 
plate  method. 

The  following  method,  suggested  by  Hazen  and  White,  has  been 
tested  with  favorable  results  by  Foote.  This  method  depends  upon 
the  fact  that  most  of  the  common  water  bacilli  do  not  grow  at  a  tem- 
perature of  40°  C.,  whereas  this  is  a  favorable  temperature  for  the 
development  of  the  typhoid  bacillus.  A  small  quantity  of  the  sus- 
pected water  is  added  to  liquefied  nutrient  agar  in  test  tubes,  and 
plates  are  made.  These  are  placed  in  an  incubating  oven  at  40°  C., 
and  the  typhoid  bacillus,  if  present,  will  develop  colonies  within  two 
or  three  days.  At  the  ordinary  room  temperature  the  more  numerous 
water  bacilli  would  develop  upon  the  same  plates  so  abundantly  that 
it  would  be  difficult  to  recognize  colonies  of  the  typhoid  bacillus. 

Theobald  Smith  (Centralb.  /.  Bakteriol.,  Bd.  xii.,  page  367), 
has  shown  that  the  typhoid  bacillus  may  be  differentiated  from  other 
similar  bacilli  (Bacillus  coli  communis,  bacillus  of  hog  cholera,  etc.) 
by  the  fact  that  it  does  not  produce  gas  in  culture  media  containing 
Hiigar—  grape  sugar,  cane  sugar,  or  milk  sugar.  The  medium  recom- 


THE   BACILLUS   OF  TYPHOID   FEVEK.  367 

mended  by  Smith  for  making  this  test  is  a  peptone-bouillon  contain- 
ing two  per  cent  of  grape  sugar  and  made  slightly  alkaline  with 
carbonate  of  soda.  The  liquid  becomes  clouded  throughout  at  the 
end  of  twenty-four  hours,  but  not  a  trace  of  gas  is  developed  even 
after  several  days.  On  the  other  hand,  the  colon  bacillus  and  other 
bacilli  which  closely  resemble  the  typhoid  bacillus  cause  an  abundant 
development  of  gas  in  this  medium. 

The  method  of  Wurtz  will  be  found  useful  in  the  detection  of 
colonies  of  the  typhoid  bacillus  in  plate  cultures  from  contaminated 
water,  etc.  This  consists  in  the  addition  to  the  nutrient  medium  of 
lactose  (two  per  cent)  and  a  solution  of  litmus.  When  the  colonies 
develop  in  plates  made  from  this  medium  the  typhoid  colonies  re- 
main blue,  while  colonies  of  the  "  colon  bacillus  "  have  a  red  color,  on 
account  of  the  development  of  lactic  acid. 

Schild  (1894)  uses  a  bouillon  containing  formalin  (1:7,000)  and 
claims  that  the  typhoid  bacillus  fails  to  grow  in  this  medium,  while 
the  bacilli  of  the  colon  group  multiply  in  it  and  cause  the  me- 
dium to  become  clouded  within  twenty-four  hours.  Abel  (1894),  as  a 
result  of  extended  experiments,  arrives  at  the  conclusion  that  the 
formalin  test  cannot  be  relied  upon  for  distinguishing  the  typhoid 
bacillus  from  certain  similar  bacilli,  which  also  fail  to  grow  in  for- 
malin solution.  But,  on  the  other  hand,  a  bacillus  which  grows  in 
bouillon  containing  1 : 7,000  of  formalin  can  be  definitely  pronounced 
to  be  not  the  typhoid  bacillus. 

Eisner  (1895)  recommends  the  following  method  for  the  detection 
of  the  typhoid  bacillus  in  water  or  in  f  aaces :  To  potato  gelatin,  pre- 
pared by  the  method  of  Holz,  he  added  one  per  cent  of  potassium 
iodide.  But  few  species  of  bacteria  will  grow  in  this  medium,  but 
Bacillus  coli  communis  grows  in  it  luxuriantly,  forming  fully  de- 
veloped colonies  at  the  end  of  twenty-four  hours.  The  typhoid  col- 
onies, on  the  contrary,  are  only  just  visible  under  a  low  power  at  the 
end  of  twenty-four  hours,  and  at  the  end  of  forty-eight  hours  are 
seen  as  small,  shining,  drop-like,  very  finely  granular  colonies.  At 
the  same  time  the  colonies  of  the  colon  bacillus  are  much  larger, 
coarsely  granular,  and  of  a  brownish  color.  By  this  method  Eisner 
succeeded  in  obtaining  pure  cultures  of  the  typhoid  bacillus  from 
the  faeces  in  fifteen  out  of  seventeen  cases  of  typhoid  fever,  in  various 
stages  of  the  disease.  Lazarus  (1895)  has  tested  this  method  and  re- 
ports that  he  succeeded  without  any  difficulty  in  obtaining  pure  cul- 
tures of  the  typhoid  bacillus  from  the  alvine  discharges  of  typhoid 
patients. 

When  the  typhoid  bacillus  and  the  colon  bacillus  are  planted  to- 
gether, in  the  same  liquid  medium,  the  first-mentioned  bacillus,  even 
when  in  excess  at  the  outset  of  the  experiment,  soon  disappears  and 


368  THE  BACILLUS   OF  TYPHOID   FEVER. 

the  Bacillus  coli  communis  remains  in  full  possession.  According 
to  Wathelet  (1895)  the  colon  bacillus  will  grow  in  bouillon  which 
has  served  as  a  culture  medium  for  the  typhoid  bacillus,  or  on  the 
surface  of  an  agar  plate  from  which  a  typhoid  culture  has  been  re- 
moved; but  the  typhoid  bacillus  fails  to  develop  in  culture  media 
which  have  served  for  the  development  of  the  colon  bacillus. 

The  various  diagnostic  tests  which  have  been  proposed,  and  the 
extensive  literature  of  the  subject,  show  that  the  recognition  of  the 
typhoid  bacillus  in  water,  faeces,  etc.,  is  attended  with  serious  diffi- 
culties. This  is  chiefly  due  to  the  fact  that  bacilli  have  been  ob- 
tained from  various  sources  which  resemble  more  or  less  closely  the 
typical  typhoid  bacillus  as  obtained  from  the  spleen  of  a  typhoid 
patient  (or  cadaver)  and  the  "  colon  bacillus  "  as  found  in  the  alimen- 
tary canal  of  healthy  men  and  animals;  and  also  from  the  fact  that 
the  bacillus,  as  obtained  from  typhoid  cases,  varies  to  some  extent  in 
its  biological  characters,  and  that  varieties  may  be  produced  in  the 
bacillus  as  obtained,  from  a  single  colony,  by  special  modes  of  culti- 
vation. From  a  consideration  of  these  facts  certain  authors  have 
been  led  to  the  conclusion  that  Bacillus  typhi  abdominalis  and  Bacillus 
coli  communis  are  simply  varieties  of  the  same  species.  This  view, 
however,  is  not  generally  accepted,  and  the  characters  which  serve  to 
differentiate  the  two  bacilli  are  sufficiently  well  defined  when  typical 
cultures  are  compared.  These  characters,  briefly  stated,  are:  The 
invisible  growth  of  the  typhoid  bacillus  on  potato ;  its  failure  to  give 
the  indol  reaction;  its  failure  to  coagulate  milk,  or  to  produce  a 
change  of  color  in  litmus  milk ;  its  failure  to  produce  gas  in  culture 
media  containing  glucose  or  lactose ;  its  failure  to  grow  in  formalin 
bouillon  (1 : 7,000) ;  and  its  active  motility.  Whether  the  closely  re- 
lated bacilli  which  present  some  of  the  characters  above  indicated, 
without  corresponding  in  all  particulars  with  typical  cultures  of  the 
typhoid  bacillus,  are  varieties  of  this  bacillus,  which  under  favorable 
circumstances  could  give  rise  to  typhoid  infection,  has  not  been  defi- 
nitely determined,  but  appears  to  be  quite  probable.  It  may  be  that 
such  varieties  are  developed  when  the  typhoid  bacillus  in  faeces  finds 
its  way  into  surface  waters,  under  conditions  which  are  favorable  for 
its  continued  development  as  a  saprophyte.  On  the  other  hand,  it 
may  be  that  one  or  more  of  the  saprophytic  bacilli,  which  are  found 
in  water  and  which  closely  resemble  the  typhoid  bacillus,  may  give  rise 
to  the  infectious  disease  which  we  know  as  typhoid  fever  when  in- 
t induced  into  the  alimentary  canal  of  a  particularly  susceptible  indi- 
vidual, and  that  the  special  conditions  attending  its  development  as 
a  parasite  give  rise  to  certain  modifications  in  its  biological  charac- 
ters of  a  nu.re  or  less  permanent  kind. 

Frankland  (18U5),  as  a  result  of  extended  experiments,  has  arrived 


THE   BACILLUS    OF   TYPHOID   FEVER.  369 

at  the  conclusion  that  when  the  typhoid  bacillus  is  cultivated  for  a 
long  time  in  media  which  are  more  and  more  largely  diluted  with 
water,  it  acquires  an  increased  ability  to  survive  in  river  water. 

A  predisposition  to  typhoid  infection  is  established  by  various 
-depressing  agencies,  such  as  inanition,  overwork,  mental  worry,  in- 
sanitary surroundings,  etc.  And  there  is  considerable  evidence  in 
support  of  the  supposition  that  exposure  to  the  offensive  gases 
given  off  from  ill-ventilated  sewers  constitutes  a  predisposition  to 
the  disease. 

Experiments  made  by  Alessi  (1894),  in  the  Hygienic  Institute 
of  the  University  of  Rome,  give  support  to  this  view.  The  ex- 
periments were  made  upon  rats,  guinea-pigs,  and  rabbits.  The 
rats  were  confined  in  a  close  cage  with  perforated  bottom,  which  was 
placed  over  the  opening  of  a  privy ;  the  guinea-pigs  and  rabbits  in 
similar  cages  having  a  receptacle  below  in  which  their  own  excreta 
was  allowed  to  accumulate.  The  animals  which  breathed  an  atmo- 
sphere vitiated  in  this  way  lost,  after  a  time,  their  usual  activity  and 
became  emaciated,  although  they  continued  to  eat  greedily.  When 
these  animals  were  inoculated  with  a  small  quantity  of  a  culture  of 
the  typhoid  bacillus  (0.25  to  0.5  cubic  centimetre)  they  died  within 
from  twelve  to  thirty-six  hours.  The  same  amount  of  the  typhoid 
culture  injected  into  control  animals  produced  no  injurious  effect.  In 
the  animals  which  succumbed  to  typhoid  infection  there  was  found  a 
hemorrhagic  enteritis,  increase  in  volume  of  Peyer's  glands  and  of  the 
spleen,  and  typhoid  bacilli  in  the  blood,  liver,  and  spleen.  The  char- 
acteristic appearances  of  typhoid  infection  were  more  pronounced  in 
the  rabbits  and  guinea-pigs  than  in  rats.  Similar  experiments  with 
Bacillus  coli  communis  gave  similar  results.  The  time  required  to 
induce  this  predisposition  for  typhoid  infection  was  from  five  to 
seventy-two  days  for  the  rats,  seven  to  fifty-eight  for  the  guinea- 
pigs,  and  three  to  eighteen  for  the  rabbits.  Alessi  found  that  the 
susceptibility  to  infection  diminished  after  a  certain  time,  and  sug- 
gests that  in  a  similar  way  man  may  become  habituated  to  breathing 
an  atmosphere  containing  sewer  gases. 

Pus- Product  ion  by  Typhoid  Bacilli. — The  recent  literature  re- 
lating to  the  typhoid  bacillus  includes  many  observations  as  to  its 
presence  in  accumulations  of  pus  in  various  parts  of  the  body — often 
in  a  pure  culture.  It  has  been  found  in  a  considerable  number  of 
cases  of  periostitis  secondary  to  typhoid  fever,  in  purulent  syno- 
vitis,  and  in  abscesses  in  various  parts  of  the  body. 

Dmochowski  and  Janowski  (1895),  as  the  result  of  a  review  of  the 
literature  and  a  painstaking  experimental  research,  arrive  at  the  con- 
clusion that  ev^n  in  abscesses,  occurring  in  typhoid  fever  cases,  in 
which  only  the  pus  cocci  are  found,  it  is  probable  that  the  typhoid 

•  %  <.  A 


370  THE   BACILLUS   OP  TYPHOID   FEVER. 

bacillus  originated  the  process  resulting  in  abscess  formation.  They 
assert  that  the  typhoid  bacillus  dies  out  in  a  comparatively  short 
time  in  abscesses  which  are  directly  due  to  its  presence,  and  that 
often  it  may  be  found  in  the  abscess  walls  when  its  presence  can  no 
longer  be  demonstrated  in  the  purulent  contents  of  the  abscess  cavity. 


PLATE  V. 

PATHOGENIC  BACTERIA. 

Fia.  1. — Bacillus  anthracis  from  cellular  tissue  of  inoculated  mouse. 
Stained  with  gentian  violet,  x  1,000.  Photomicrograph  by  Frankel  and 
Pfeiffer. 

FIG.  2. — Bacillus  anthracis  in  section  of  liver  of  inoculated  rabbit. 
Stained  with  Bismarck  brown,  x  250.  Photomicrograph  by  Sternberg. 

Fia.  3. — Micrococcus  gonorrhoeas  in  gonorrhoeal  pus.  Stained  with  gen- 
tian violet,  x  1,000.  Photomicrograph  by  gaslight  (Sternberg). 

FlG.  4. — Anthrax  spores  from  a  bouillon  culture.  Double-stained  prepara- 
tion— with  carbol-fuchsin  and  methylene  blue,  x  1,000.  Photomicrograph 
by  Frankel  and  Pfeiffer. 

FIG.  5. — Spirillum  choleras  Asiaticae  from  a  culture  upon  starched  linen 
at  end  of  twenty-four  hours  Stained  with  fuchsin.  x  1,000.  Photomi- 
crograph by  Frankel  and  Pfeiffer. 

FIG.  6.— Bacillus  diphtherias  from  colony  upon  -an  agar  plate,  twenty- 
four  hours  old.  Stained  with  Ltf  filer's  solution  of  methylene  blue,  x  1,000. 
Photomicrograph  by  Friinkel  and  Pfeiffer. 


Mi  II-.    -••  ,:,. 


PLATH  V. 

STERNBERG'S  BACTERIOLOGY 


, 


,. 


»  . 


I'1  IK. 


PATHOGENIC  BACTERIA 


IX. 
BACTERIA  IN  DIPHTHERIA. 

DIPHTHERIA  is  generally  recognized  by  physicians  as  a  specific 
infectious  disease,  and,  owing  to  its  wide  prevalence  and  fatal  char- 
acter, a  precise  knowledge  of  its  etiology  is  of  the  greatest  import- 
ance. Until,  as  a  result  of  recent  researches,  this  was  determined, 
pathologists  were  in  doubt  as  to  whether  diphtheria  should  be  con- 
sidered as  primarily  a  local  infection,  or  whether  the  local  manifesta- 
tions were  secondary  to  a  general  systemic  infection.  But  this  question 
appears  now  to  be  definitely  settled  in  favor  of  the  former  view.  We 
have  to-day  a  very  precise  knowledge  of  the  specific  infecting  agent, 
and  have  evidence  that  it  produces  during  its  growth  a  very  potent 
toxic  substance,  the  absorption  of  which  from  the  seat  of  local  infec- 
tion accounts  in  a  satisfactory  manner  for  the  general  symptoms  of 
the  disease,  which  are  due  to  toxaemia  and  not  to  an  invasion  of  the 
blood  and  tissues  by  the  pathogenic  microorganism  producing  it. 

Numerous  researches  by  competent  bacteriologists  have  failed  to 
demonstrate  the  presence  of  bacteria  in  the  blood  of  patients  suffer- 
ing from  diphtheria,  but  a  variety  of  microorganisms  have  been  ob- 
tained in  cultures  from  diphtheritic  pseudo-membranes,  and  may  be 
demonstrated  by  the  microscopical  examination  of  stained  prepara- 
tions. Among  these  are  the  well-known  pus  organisms,  and  espe- 
cially the  Streptococcus  pyogenes,  which  appears  to  be  very  commonly 
present,  and  is  perhaps  the  active  agent  in  the  production  of  certain 
forms  of  pseudo-diphtheria.  But  the  malignant,  specific  diphtheria, 
so  well  known  in  this  country  and  in  Europe,  has  been  demonstrated 
by  the  recent  researches  of  bacteriologists  to  be  due  to  a  bacillus  first 
recognized  by  Klebs  in  stained  preparations  of  diphtheritic  false 
membranes  (1883),  and  cultivated  and  described  by  Loffler  in  1884. 
In  his  first  publication  Loffler  did  not  claim  to  have  fully  demon- 
strated the  etiological  relation  of  this  bacillus,  but  this  appears  to  be 
fully  established  by  subsequent  researches. 

In  his  first  research  Loftier  studied  twenty-five  cases,  and  in  the 
greater  number  of  them  found  in  stained  preparations  the  bacil- 
lus previously  described  by  Klebs.  From  six  of  these  cases  he 


372  BACTERIA  IN  DIPHTHERIA. 

obtained  it  in  pure  cultures,  and  by  inoculations  in  pigeons,  chickens, 
rabbits,  and  guinea-pigs  proved  that  it  gave  rise  to  a  diphtheritic 
inflammation  when  inoculated  into  the  mucous  membrane  of  the 
trachea,  conjunctiva,  pharynx,  or  vagina.  In  a  second  communica- 
tion Loffler  reported  his  success  in  finding  the  same  bacillus  in  ten 
additional  cases,  and  also  that  he  had  isolated  from  the  same  source 
a  non-pathogenic  bacillus  which  resembled  it  very  closely.  This 
pseudo-diphtheria  bacillus  has  since  been  found  by  other  bacteri- 
ologists (Von  Hoffmann,  Roux  and  Yersin),  and  it  is  uncertain 
whether  it  is  to  be  considered  a  distinct  species,  or  a  non-pathogenic 
variety  of  the  diphtheria  bacillus  as  maintained  by  Roux  and  Yersin. 
But  its  occasional  presence  does  not  invalidate  the  very  positive  ex- 
perimental evidence  relating  to  the  specific  pathogenic  power  of  the 
true  diphtheria  bacillus. 

Loffler,  in  1890,  reviewed  the  evidence  upon  which  this  bacillus  is 
now  generally  conceded  by  bacteriologists  to  be  the  specific  infectious 
agent  in  true  diphtheria.  The  following  are  the  principal  points  in 
the  demonstration : 

FIRST. — It  is  found  in  all  undoubted  cases  of  diphtheria.  In 
support  of  this  we  have  the  results  of  researches  made  by  Loffler, 
Wyssokowitsch,  D'Espine,  Yon  Hoffmann,  Ortmann,  Roux  and 
Yersin,  Kolisko  and  Paltauf,  Zarinko  and  Sorensen,  who  in  nearly 
every  case  have  demonstrated  without  difficulty  the  presence  of  this 
bacillus.  On  the  other  hand,  Prudden  failed  to  find  it  in  a  series  of 
twenty-four  cases  studied  by  him ;  but  his  own  account  of  these 
cases  indicates  that  they  were  not  cases  of  true  diphtheria.  He  says 
in  a  subsequent  communication  : 

"  In  view  of  the  doubt  existing  among  practitioners  as  to  whether  all 
forms  of  pseudo-membranous  inflammation  should  be  called  diphtheria  or 
not,  and  with  the  purpose  of  making  a  wholly  objective  study,  the  writer 
distinctly  stated  at  the  outset  of  that  paper  that  all  the  fatal  cases  of  exten- 
sive pseudo-membranous  laryngitis,  as  well  as  pharyngitis,  should  in  his 
study  be  considered  as  cases  of  diphtheria.  This  left  the  question  as  to  the 
propriety  of  establishing  separate  groups  of  pseudo-membranous  inflamma- 
tion open  and  free  from  bias.  It  was  distinctly  stated,  however,  that  six- 
teen out  of  the  twenty-four  cases  occurred  in  a  large  asylum,  in  which 
measles  and  scarlet  fever  were  prevalent  during  the  period  in  which  these 
studies  were  under  way.  Five  other  cases  in  another  asylum  were  ex- 
posed to  similar  conditions." 

In  a  subsequent  series  of  "twelve  cases  of  fatal  pseudo-mem- 
branous inflammation  occurring  in  two  children's  asylums,  in  which 
t  T  many  months  there  had  been  no  scarlatina  and  no  measles,  and 
in  which  there  was  no  complicating  suppurative  inflammation  and 
ii"  <  i  \ -sipelas,"  Prudden  (1890)  obtained  Loffler's  bacillus  in  cultures 
t  i"in  Cloven,  and  he  says  : 

"  We  are  now,  it  would  seem,  justified,  as  it  did  not  appear  to  the  writer 


BACTERIA   IN   DIPHTHERIA.  373 

that  we  were  two  years  ago,  owing  to  the  large  number  of  important  re- 
searches which  have  been  made  in  the  interim,  in  saying  that  the  name 
diphtheria,  or  at  least  primary  diphtheria,  should  be  applied,  and  exclusively 
applied,  to  that  acute  infectious  disease,  usually  associated  with  a  pseudo- 
membranous  inflammation  of  the  mucous  membranes,  which  is  primarily 
caused  by  the  bacillus  called  Bacillus  diphtherias  of  Loftier." 

With  reference  to  the  question  as  to  how  long  after  convalescence 
is  established  the  diphtheria  bacillus  may  be  present  in  the  throat 
of  an  infected  person,  Loffler  has  made  the  following  research  (1890). 
In  a  typical  case  a  bacteriological  examination  was  made  daily  from 
the  commencement  until  fourteen  days  after  its  termination.  Fever 
disappeared  on  the  fifth  day,  and  the  exudation  had  all  disappeared 
on  the  sixteenth  day.  Up  to  this  time  the  bacillus  was  daily  ob- 
tained in  cultures,  and  subsequently  nearly  every  day  up  to  the 
twenty-fifth — that  is,  for  three  weeks  after  the  febrile  symptoms  had 
disappeared.  Roux  and  Yersin  have  also  obtained  the  bacillus  in 
cultures  from  mucus  scraped  from  the  throats  of  convalescents  sev- 
eral days  after  the  disappearance  of  all  evidence  of  the  disease. 

SECOND.  The  Klebs-Loffler  bacillus  is  found  only  in  diph- 
theria.— In  his  earlier  researches  Loffler  obtained  the  bacillus  in  a 
single  instance  from  the  mouth  of  a  healthy  child,  and  this  fact  led 
him  to  hesitate  in  announcing  it  as  his  conviction  that  it  was  the 
true  cause  of  diphtheria.  But  in  extended  researches  made  subse- 
quently he  has  not  again  succeeded  in  finding  it,  except  in  associa- 
tion with  diphtheria,  and  admits  now  that  he  may  have  been  mis- 
taken as  to  the  identity  of  the  bacillus  found.  This  seems  not 
improbable  in  view  of  the  fact  that  very  similar  bacilli  have  been 
found  by  various  bacteriologists.  Thus  Von  Hoffmann  obtained  a 
very  similar  but  non-pathogenic  bacillus  from  the  mucus  of  chronic 
nasal  catarrh  and  from  healthy  mucous  membranes ;  Babes  from 
cases  of  trachoma,  Neisser  from  ulcers,  Zarinko  rrom  the  surface  of 
various  mucous  membranes.  But  all  of  these  were  shown  to  present 
certain  differences  in  their  biological  characters  by  which  they  could 
be  differentiated  from  the  true  diphtheria  bacillus. 

Welch  and  Abbott  in  their  comparative  studies  did  not  find  the 
Loffler  bacillus,  "or  any  bacillus  that  an  experienced  bacteriologist 
would  be  likely  to  confound  with  it."  They  examined  mucus  from 
the  throats  of  healthy  children,  from  those  suffering  from  simple  in- 
flammation of  the  tonsils  and  pharynx,  and  from  four  cases  of  so- 
called  follicular  tonsillitis.  As  a  result  of  their  investigations  they 
agree  with  Loffler,  and  with  Roux  and  Yersin,  as  to  "  the  great  prac- 
tical value,  for  diagnostic  purposes,  of  a  bacteriological  examination 
of  cover-glass  specimens  and  by  cultures  "  of  cases  in  which  there  is 
any  doubt  of  the  true  character  of  the  disease.  They  say  further  : 


374  BACTERIA  IN   DIPHTHERIA. 

"The  only  species  of  bacteria  which  we  have  found  constantly  in  the 
cases  of  diphtheria  has  been  the  Loftier  bacillus.  Two  other  species  have 
been  present  in  many  cases,  viz.,  the  well-known  streptococcus,  which  grows 
in  much  smaller  colonies  and  less  rapidly  than  the  Loftier  bacillus,  and  a 


TlSe  colonies  of  this  bacillus  are  grayish-white,  moist,  larger  than  those  of 
the  streptococcus,  but  smaller  than  those  of  the  Loftier  bacillus." 

THIRD.  As  shown  by  Ldffler>s  earlier  researches,  pure  cultures 
of  this  bacillus  induce  characteristic  diphtheritic  inflammation 
when  inoculated  into  the  mucous  membranes  of  certain  lower  ani- 
mals. Roux  and  Yersin  have  also  shown  that  local  paralysis  is 
likely  to  occur  in  inoculated  animals,  as  is  the  case  in  diphtheria  in 
man.  In  speaking  of  their  inoculations  into  the  trachea  in  rabbits 
those  investigators  say  : 

"The  affection  which  is  thus  induced  in  the  rabbit  resembles  croup  in 
man.  The  difficulty  which  the  animal  experiences  in  breathing ;  the  noise 
made  by  the  air  in  passing  through  the  obstructed  trachea-  the  aspect  of  the 
trachea,  which  is  congested  and  covered  with  false  membranes ;  the  cedema- 
tous  swelling  of  the  tissues  and  glands  of  the  neck,  make  the  resemblance 
absolutely  remarkable." 

Welch  and  Abbott  give  the  following  account  of  the  results  of 
inoculations  into  the  trachea  in  kittens  : 

"A  half-grown  kitten  is  inoculated  into  the  trachea  with  one  platinum 
loop  from  a  pure  culture  of  the  Loftier  bacillus  on  glycerin-a^ar,  eleven  days 
old,  derived  from  Case  IV.  For  the  inoculation  a  small  median  incision  was 
made  over  the  trachea,  in  which  a  hole  just  large  enough  to  admit  the  plati- 
num loop  was  made.  The  culture  was  rubbed  over  the  mucosa  of  the  trachea 
for  an  extent  about  three  centimetres  in  length,  and  in  this  process  sufficient 
force  was  used  to  abrade  the  mucous  membrane.  On  the  day  following  the 
inoculation  no  special  alteration  in  the  animal  was  observed,  but  on  the 
morning  of  the  second  day  it  was  found  very  weak.  In  the  course  of  this 
day  it  became  so  weak  as  to  lie  completely  motionless,  apparently  uncon- 
scious, with  very  feeble,  shallow  respiration;  several  times  it  was  thought  to 
be  dead,  but  on  careful  examination  proved  still  to  be  breathing  feebly.  It 
was  found  dead  on  the  morning  of  the  third  day.  At  the  autopsy  the  wound 
w.ts  found  gaping  and  covered  with  a  grayish,  adherent,  necrotic,  distinctly 
diphtheritic  layer.  For  a  considerable  distance  around  the  wound  the  sub- 
cutaneous tissues  were  very  cedematous,  the  oedema  extending  from  the 
lower  jaw  down  over  the  sternum,  and  to  the  sides  of  the  neck,  and  along 
the  anterior  extremities.  The  lymphatic  glands  at  the  angle  of  the  jaw  were 
markedly  swollen  and  reddened.  The  mucous  membrane  of  the  trachea, 
Ix'^mmiitfat  th<-  l.-irynxand  extending  down  forsix  centimetres,  wascovered 
with  a  tolerably  firm,  grayish-white,  loosely  attached  pseudo-membrane,  in 
;ill  respects  identical  with  the  croupous  membranes  observed  in  the  same 
situation  in  cases  of  human  diphtheria." 


BACTERIA   IN  DIPHTHERIA.  375 

47.    BACILLUS   DIPHTHERIA. 

First  observed  by  Klebs  (1883)  in  diphtheritic  false  membranes. 
Isolated  in  pure  cultures  and  pathogenic  power  demonstrated  by 
Loffler  (1884). 

Found  in  diphtheritic  pseudo-membranes,  and  especially  in  the 
deeper  portions,  intermingled  with  numerous  cellular  elements;  while 
the  superficial  layers  of  the  membrane  commonly  contain  but  few 
cells  or  bacilli,  or  are  invaded  by  other  species,  especially  by  Strep- 
tococcus pyogenes.  The  bacilli  are  not  found  in  the  affected  mucous 
membrane,  or  in  sections  from  the  internal  organs  in  fatal  cases  of 
this  disease. 

Morphology. — Rods,  straight  or  slightly  curved,  with  rounded 
ends,  having  a  diameter  of  0.5  to  0.8 
jt,  and  from  2  to  3  IJL  in  length.  Ir- 
regular forms  are  very  common,  and, 
indeed,  are  characteristic  of  this  bacil- 
lus. In  the  same  culture,  and  especially 
in  an  unfavorable  culture  medium,  very 
great  differences  in  form  and  dimen- 
sions may  be  observed  ;  one  or  both  ends 
may  appear  swollen,  or  the  central  por- 
tion may  be  notably  thicker  than  the 
extremities,  or  the  rod  may  be  made  up 

of  irregular  spherical  or  oval  segments.     FIG.  114.  —  Bacillus  diphtheria*, 
Multiplication  occurs  by  fission  only,  SLtJSlS 

aild  the  bacilli  do  not  grOW  Out  into  fila-    (Frankel  and  Pfeiffer.) 

ments. 

In  unstained  preparations  certain  portions  of  the  rod,  and  espe- 
cially the  extremities,  are  observed  to  be  more  highly  refractive  than 
the  remaining  portion  ;  and  in  stained  preparations  these  portions 
are  seen  to  be  most  deeply  colored.  The  diphtheria  bacillus  may  be 
stained  by  the  use  of  Loffler's  alkaline  solution  of  methylene  blue, 
but  is  not  so  readily  stained  with  some  of  the  other  aniline  colors 
commonly  employed.  It  stains  also  by  Gram's  method.  For  the 
demonstration  of  the  bacillus  in  sections  of  diphtheritic  membrane 
"  nothing  can  surpass  in  brilliancy  and  sharp  differentiation  sections 
stained  doubly  by  the  modified  Weigert's  fibrin  stain  and  picro-car- 
mine"  (Welch  and  Abbott). 

Biological  Characters. — The  diphtheria  bacillus  is  aerobic,  non- 
motile,  and  non-liquefying;  it  does  not  form  spores.  It  grows  most 
freely  in  the  presence  of  oxygen,  but  is  also  a  facultative  anaerobic. 

Development  occurs  in  various  culture  media  at  a  temperature  of 
from  20°  to  42°  C.,  the  most  favorable  temperature  being  about  35°  C. 


BACTERIA   IX   DIPHTHERIA. 

It  grows  readily  in  nutrient  gelatin  having  a  slightly  alkaline  reac- 
tion, in  nutrient  agar,  glycerin-agar,  or  in  alkaline  bouillon,  but  the 
most  favorable  medium  appears  to  be  that  first  recommended  by 

Loffler—  viz.,  a  mixture  of  three 
parts  of  blood  serum  with  one  part 
of  bouillon,  containing  one  per  cent 
of  peptone,  one  per  cent  of  grape 
sugar,  and  0.5  per  cent  of  sodium 
chloride.  This  mixture  is  steril- 
ized and  solidified  at  a  low  tem- 
perature, as  is  usual  with  blood 
serum.  Upon  this  the  develop- 
FIO.  ns.-coionies  of  Bacillus  diphtheria  inent  is  so  rapid  in  the  incubating 


in  nutrient  a&ar,  end  of  twenty-four  hours.     oven   ^hat,  ^   ^he   end    of  twentv- 
X  10.    (Frankel  and  Pfeiffer.)  ,11  j        i 

four  hours,  the  large,  round,  ele- 

vated colonies,  of  a  grayish-  white  color  and  moist  appearance,  may 
be  easily  recognized,  while  other  associated  bacteria  will,  as  a  rule, 
not  yet  have  developed  colonies  large  enough  to  interfere  with  the- 
recognition  of  these. 

Upon  nutrient  agar  plates  the  deep-lying  colonies,  when  magni- 

fied about  eighty  diameters,  appear  as  round  or  oval,  coarsely  granu- 

lar discs,  with  rather  ill-defined  margins,  or,  when  several  colonies 

are  in  juxtaposition,  as  figures  of  irregular  form.     The  superficial  col- 

onies are  grayish-yellow  in  color,  have  an  irregular,  not  well-defined 

outline  and  a  rough,  almost  reticulated  surface.     The  growth  upon 

glycerin-agar  is  very  similar.     The  first  inoculations  in  a  plain  nu- 

trient agar  tube  often  give  a  comparatively  feeble  growth,  which  be- 

comes more  abundant  in  subsequent  inoculations  in  the  same  medium. 

In  stick  cultures  in  glycerin  —  or  plain  —  agar,  growth  occurs  to  the 

bottom  of  the  line  of  inoculation,  and  also  upon  the  surface,  but  is 

not  at  all  characteristic.     The  same  may  be  said  with  reference  to 

cultures  in  nutrient  gelatin.     Plate  cultures  in  this  medium  contain- 

ing fifteen  per  cent  of  gelatin,  at  24°  C.,  give  rather  small  colonies, 

which  are  white  by  reflected  light  and  under  the  microscope  are  seen 

as  yellowish-brown,  opaque  discs,  having  a  more  or  less  irregular 

outline  and  a  granular  structure.     In  alkaline  bouillon  the  growth  is 

sometimes  in  the  form  of  small,  whitish  masses  along  the  sides  and 

bottom  of  the  tube,  but  at  others  a  diffusely  clouded  growth  occurs 

in  this  medium  ;  after  standing  for  some  time  in  the  incubating  oven 

a  thin,  white  pellicle  may  form  upon  the  surface  of  the  bouillon. 

'I'  1  10  reaction  of  the  bouillon  becomes  at  first  acid,  but  later  it  has  an 

alkaline  reaction  (Welch).     With  reference  to  the  growth  onpotato? 

authors  have  differed,  probably  because  the  growth  is  scarcely  vis- 

ible ;  upon  this  point  we  quote  from  Welch  and  Abbott  : 


BACTERIA  IN   DIPHTHERIA.  377 

"  Our  experience  lias  been  that  the  Bacillus  diphtherias  grows  on  ordinary 
steamed  potato  without  any  preliminary  treatment,  but  that  the  growth  is 
usually  entirely  invisible  or  is  indicated  by  a  dry,  thin  glaze  after  several 
days.  Doubtless  the  invisible  character  of  the  growth  has  led  most  observers 
into  the  error  of  supposing  that  no  growth  existed,  whereas  the  microscopi- 
cal examination  reveals  a  tolerably  abundant  growth,  which  on  the  first  po- 
tato is  often  feebler  than  on  succeeding  ones.  Irregular  forms  are  par- 
ticularly numerous  in  potato  cultures,  and  in  general  the  rods  are  thicker 
than  on  other  media.  In  twenty-four  hours,  at  a  temperature  of  35°  C., 
microscopical  examination  shows  distinct  growth.  We  have  cultivated  the 
bacillus  for  many  generations  on  potato." 

Milk  is  a  favorable  medium  for  the  growth  of  this  bacillus,  and, 
as  it  grows  at  a  comparatively  low  temperature  (20°  C.),  it  is  evi- 
dent that  this  fluid  may  become  a  medium  for  conveying  the  bacillus 
from  an  infected  source  to  the  throats  of  previously  healthy  children. 

Cultures  of  the  diphtheria  bacillus  may  retain  their  vitality  for 
several  months,  and  when  dried  upon  silk  threads  for  several  weeks 
colonies  are  still  developed  in  a  suitable  medium — in  the  room  from 
three  to  four  weeks,  in  an  exsiccator  five  to  ten,  and  in  one  instance 
fourteen  weeks.  In  dried  diphtheritic  membrane,  preserved  in  small 
fragments,  the  bacillus  retained  its  vitality  for  nine  weeks,  and  in 
larger  fragments  for  twelve  to  fourteen  weeks. 

The  thermal  death-point,  as  determined  by  Welch  and  Abbott,  is 
58°  C. ,  the  time  of  exposure  being  ten  minutes.  Loffler  had  previ- 
ously found  that  it  did  not  survive  exposure  for  half  an  hour  to  60° 
C.  With  reference  to  the  action  of  germicidal  and  antiseptic  agents, 
we  refer  to  the  sections  in  Part  Second  relating  to  this  subject. 

Pathogenesis. — In  view  of  the  evidence  heretofore  recorded,  it 
may  be  considered  as  demonstrated  that  this  bacillus  gives  rise  to 
the  morbid  phenomena  which  characterize  the  fatal  disease  in  man 
known  as  diphtheria. 

We  have  already  referred  to  the  effects  of  inoculations  into  the 
trachea  in  rabbits  and  cats,  which  give  rise  to  a  characteristic  diph- 
theritic inflammation,  with  general  toxaemia  and  death  from  the 
absorption  of  soluble  toxic  products  formed  at  the  seat  of  local  in- 
fection. This  inference  as  to  the  cause  of  death  seems  justified  by 
the  fact  that  the  pathogenic  bacillus  does  not  invade  the  blood  and 
tissues,  and  is  supported  by  additional  experimental  evidence  which 
we  give  below.  Subcutaneous  inoculations  in  guinea-pigs  of  a  small 
quantity  of  a  pure  culture  of  the  bacillus  (0. 1  to  0. 5  cubic  centime- 
tre of  a  bouillon  culture)  cause  death  in  from  one  to  four  or  five 
days.  The  usual  changes  observed  at  the  autopsy  are  "  an  exten- 
sive local  oedema  with  more  or  less  hypersemia  and  ecchymosis  at 
the  site  of  inoculation,  frequently  swollen  and  reddened  lymphatic 
glands,  increased  serous  fluid  in  the  peritoneum,  pleura,  and  pericar- 
dium, enlarged  and  hsBmorrhagic  suprarenal  capsules,  occasionally 


378  BACTERIA   IN   DIPHTHERIA. 

slightly  swollen  spleen,  sometimes  fatty  degenerations  in  the  liver, 
kidney,  and  myocardium.  We  have  always  found  the  Loffler  ba- 
cilli at  the  seat  of  inoculation,  most  abundant  in  a  grayish-white, 
fibrino-purulent  exudate  present  at  the  point  of  inoculation,  and  be- 
coming fewer  at  a  distance  from  this,  so  that  the  more  remote  parts 
of  the  oedematous  fluid  do  not  contain  any  bacilli  "  (Welch  and  Ab- 
bott). The  authors  quoted  agree  with  Loffler  and  others  in  stating 
that  the  bacillus  is  only  found  at  the  point  of  inoculation.  In  all 
cases  their  cultures  from  the  blood  and  from  the  various  organs  gave 
a  negative  result. 

Rabbits  are  not  so  susceptible  and  may  recover  after  the  subcu- 
taneous inoculation  of  very  small  doses,  but  usually  die  in  from  four 
to  twenty  days  when  two  to  four  cubic  centimetres  of  a  bouillon 
culture  have  been  introduced  beneath  the  skin.  In  these  animals 
also  there  is  an  extensive  local  oedema,  enlargement  of  the  neigh- 
boring lymphatic  glands,  and  a  fatty  degeneration  of  the  liver. 
Roux  and  Yersin  have  shown  that  in  these  animals,  when  death 
does  not  ensue  too  quickly,  paralysis  of  the  posterior  extremities  fre- 
quently occurs,  thus  completing  the  experimental  proof  of  the  spe- 
cific pathogenic  power  of  pure  cultures  of  this  bacillus. 

Similar  symptoms  are  produced  in  pigeons  by  the  subcutaneous 
inoculation  of  0. 5  cubic  centimetre  or  more,  but  they  commonly  re- 
cover when  the  quantity  is  reduced  to  0.2  cubic  centimetre  (Roux 
and  Yersin). 

The  rat  and  the  mouse  have  a  remarkable  immunity  from  the 
effects  of  this  poison.  Thus,  according  to  Roux  and  Yersin,  a  dose 
of  two  cubic  centimetres,  which  would  kill  in  sixty  hours  a  rabbit 
weighing  three  kilogrammes,  is  without  effect  upon  a  mouse  which 
weighs  only  ten  grammes. 

Old  cultures  are  somewhat  less  virulent  than  fresh  ones,  but  when 
replanted  ia  a  fresh  culture  medium  they  manifest  their  original 
virulence.  Thus  a  culture  upon  blood  serum  which  was  five  months 
old  was  found  by  Roux  and  Yersin  to  kill  a  guinea-pig  in  five  days, 
but  when  replanted  it  killed  a  second  animal  of  the  same  species  in 
twenty-four  hour-. 

Evidently  a  microorganism  which  destroys  the  life  of  a  suscepti- 
ble animal  when  injected  beneath  its  skin  in  small  quantity,  and 
which  nevertheless  is  only  found  in  the  vicinity  of  the  point  of  in- 
oculation,  must  owe  its  pathogenic  power  to  the  formation  of  some 
pot  i -nt  toxic  substance,  which  being  absorbed  gives  rise  to  toxaemia 
•  ni.i  death.  This  inference  in  the  case  of  the  diphtheria  bacillus  is 
fully  sustained  by  the  results  of  recent  experimental  investigations. 
Roux  and  Yersin  (1888)  first  demonstrated  the  pathogenic  power  of 
cultuivs  which  had  IHVM  filtered  through  porous  porcelain.  Old 


BACTERIA   IN   DIPHTHERIA.  379 

cultures  were  found  by  these  experimenters  to  contain  more  of  the 
toxic  substance  than  recent  ones,  and  to  cause  the  death  of  a  guinea- 
pig  in  the  dose  of  two  cubic  centimetres  in  less  than  twenty-four 
hours.  The  filtered  cultures  produced  in  these  animals  the  same 
effects  as  those  containing  the  bacilli — local  oedema,  hsemorrhagic 
congestion  of  the  organs,  effusion  into  the  pleural  cavity.  Some- 
what larger  doses  were  fatal  to  rabbits,  and  a  few  drops  injected 
subcutaneously  sufficed  to  kill  a  small  bird  within  a  few  hours.  In 
their  second  paper  (1889)  the  authors  mentioned  state  that  so  long  as 
the  reaction  of  a  culture  in  bouillon  is  acid  its  toxic  power  is  com- 
paratively slight,  but  that  in  old  cultures  the  reaction  is  alkaline, 
and  in  these  the  toxic  potency  is  greatly  augmented.  With  such  a 
culture,  filtered  after  having  been  kept  for  thirty  days,  a  dose  of 
one-eighth  of  a  cubic  centimetre,  injected  subcutaneously,  sufficed 
to  kill  a  guinea-pig ;  and  in  larger  amounts  it  proved  to  be  fatal 
to  dogs  when  injected  directly  into  the  circulation  through  a  vein. 

The  same  authors,  in  discussing  the  nature  of  the  poison  in  their 
filtered  cultures,  infer  that  it  is  related  to  the  diastases,  and  state 
that  its  toxic  potency  is  very  much  reduced  by  exposure  to  a  com- 
paratively low  temperature — 58°  C.  for  two  hours — and  completely 
destroyed  by  the  boiling  temperature — 100°  for  twenty  minutes.  It 
was  found  to  be  insoluble  in  alcohol,  arid  the  precipitate  obtained  by 
adding  alcohol  to  an  old  culture  proved  to  contain  the  toxic  sub- 
stance. Loffler  also  has  obtained,  by  adding  five  volumes  of  alco- 
hol to  one  of  a  pure  culture,  a  white  precipitate,  soluble  in  water, 
which  killed  rabbits  in  the  dose  of  0. 1  to  0. 2  gramme  when  injected 
beneath  the  skin  of  these  animals.  It  gave  rise  to  a  local  oedema 
and  necrosis  of  the  skin  in  the  vicinity  of  the  point  of  inoculation, 
and  to  hypersemia  of  the  internal  organs.  This  deadly  toxin  appears 
to  be  an  albuminoid  substance,  but  its  exact  chemical  composition 
has  not  yet  been  determined.  * 

Brieger  and  Frankel  have  succeeded  in  rendering  guinea-pigs 
immune  against  virulent  cultures  of  the  diphtheria  bacillus  by  inject- 
ing bouillon  cultures  three  weeks  old,  which  had  been  sterilized  by 
exposure  for  an  hour  to  60°  to  70°  C.,  into  the  subcutaneous  tissues 
(ten  to  twenty  cubic  centimetres).  At  first  the  susceptibility  of  the 
animal  is  rather  increased  than  diminished,  but  at  the  end  of  two 
weeks  immunity  is  said  to  be  complete.  Frankel  is  of  the  opinion 
that  immunity  results  from  the  introduction  of  a  substance  which  is 
not  identical  with  the  toxic  product  to  which  the  cultures  owe  their 
pathogenic  power.  This  latter  is  destroyed  by  a  temperature  of  55° 
to  60°  C.,  while  the  substance  which  gives  immunity  is  still  present 
in  the  cultures  after  exposure  to  a  temperature  of  60°  to  70°,  as  shown 
by  the  protective  results  of  inoculations  made  with  such  cultures. 


380  BACTERIA   IN   DIPHTHERIA. 

The  researches  of  Behring  show  that  the  blood  of  immune  ani- 
mals contains  a  substance  which  neutralizes  the  toxic  product  con- 
tained in  virulent  cultures  of  the  diphtheria  bacillus.  This  effect  is 
said  to  be  produced  when  blood  from  such  an  animal  is  added  to  a 
filtered  culture  without  the  body,  as  well  as  when  the  culture  is  in- 
jected into  the  living  animal.  This  remarkable  discovery  has  al- 
ready been  utilized  for  the  treatment  of  diphtheria  in  man  with  most 
brilliant  results.  The  method  of  preparing  the  diphtheria  "anti- 
toxin "  is  given  in  the  writer's  recent  work  on  "  Immunity,  Pro- 
tective Inoculations,  and  Serum-Therapy." 

According  to  Roux  and  Yersin,  "  attenuated  varieties  "  of  the 
diphtheria  bacillus  may  be  obtained  by  cultivating  it  at  a  temperature 
of  30.5°  to  40°  C.  in  a  current  of  air  ;  and  these  authors  suggest  that 
a  similar  attenuation  of  pathogenic  power  may  occur  in  the  fauces  of 
convalescents  from  the  disease,  and  that  possibly  the  similar  non- 
pathogenic  bacilli  which  have  been  described  by  various  investiga- 
tors have  originated  in  this  way  from  the  true  diphtheria  bacillus. 
These  authors  further  state,  in  favor  of  this  view,  that  from  diphtheri- 
tic false  membrane,  preserved  by  them  in  a  desiccated  condition  for 
five  months,  they  obtained  numerous  colonies  of  the  bacillus  in  ques- 
tion, but  that  the  cultures  were  destitute  of  pathogenic  virulence. 
They  say: 

"  It  is  then  possible,  by  commencing  with  a  virulent  bacillus  of 
diphtheria,  to  obtain  artificially  a  bacillus  without  virulence,  quite 
similar  to  the  attenuated  bacilli  which  may  be  obtained  from  a  benign 
diphtheritic  angina,  or  even-  from  the  mouth  of  certain  persons  in 
good  health.  This  microbe,  obtained  artificially,  resembles  com- 
pletely the  pseudo-diphtheritic  bacillus  ;  like  it,  it  grows  more  abun- 
dantly at  a  low  temperature;  it  renders  bouillon  more  rapidly  alkaline; 
it  grows  with  difficulty  in  the  absence  of  oxygen." 
% 

48.    PSEUDO-DIPHTHERITIC   BACILLUS. 

Loffler,  Von  Hoffmann,  and  others  have  reported  finding  bacilli 
which  closely  resemble  the  Bacillus  diphtherias,  but  which  differ 
IVoiu  it  chiefly  in  being  non-pathogenic.  The  following  account  we 
take  from  the  latest  paper  upon  the  s-ubject  by  Roux  and  Yersin 
1 1  roisieme  memoire,  1800). 

Found  by  Roux  and  Yersin  in  mucus  from  the  pharynx  and  ton- 
sils of  children— from  forty-five  children  in  Paris  hospitals,  suffering 
I'n.m  various  affections,  not  diphtheritic,  fifteen  times;  from  fifty- 
nine  healthy  children  in  a  villa-o  school  on  the  seaboard,  twenty-six 
times.  Of  six  children  with  a  simple  angina  but  two  furnished  cul- 
farea  of  this  bacillus,  while  it  was  obtained  in  fiveoutof  seven  casea 
of  measles. 


BACTERIA   IN   DIPHTHERIA.  33^ 

Its  characters  are  given  as  follows  : 

"The  colonies  of  the  pseudo -diphtheritic  bacillus,  cultivated  upon  blood 
serum,  are  identical  with  the  true  diphtheria  bacillus.  At  a  temperature  of 
33°  to  35°  multiplication  is  rapid,  and  it  continues  at  the  ordinary  tempera- 
ture, although  slowly.  Under  the  microscope  the  appearance  of  the  bacillus 
which  forms  these  colonies  is  the  same  as  that  of  Bacillus  diphtherise.  It 
stains  readily  with  Loffler's  solution  of  methylene  blue,  and  intensely  by 
Gram's  method.  Sometimes  it  colors  uniformly,  at  others  it  appears  granu- 
lar. It  grows  in  alkaline  bouillon,  giving  a  deposit  upon  the  walls  of  the 
vessel  containing  the  culture,  and  in  this  medium  often  presents  the  inflated 
forms,  pear-shaped,  or  club-shaped.  It  is  destroyed  in  a  liquid  medium  by  a 
temperature  of  58°  C.  maintained  for  ten  minutes.  All  of  these  characters 
are  common  to  the  pseudo-diphtheritic  bacillus  and  the  true  Bacillus  diphthe- 
rias. As  a  difference  between  them  we  may  note  that  the  pseudo  diphtheritic 
bacillus  is  of  ten  shorter  in  colonies  grown  upon  blood  serum;  that  its  cultures 
in  bouillon  are  more  abundant ;  that  they  continue  at  a  temperature  of  20°  to 
22,  at  which  the  true  bacillus  grows  very  slowly.  When  we  make  a  com- 
parison of  cultures  in  bouillon  they  become  acid  and  then  alkaline,  but  the 
change  occurs  much  sooner  in  the  case  of  the  pseudo-diphtheritic  bacillus. 
Like  the  true  bacillus,  the  pseudo- diphtheritic  grows  in  a  vacuum,  but  less 
abundantly  than  the  other. 

k  4  Inoculations  into  animals  of  cultures  of  this  bacillus  have  never  caused 
their  death ;  but  we  may  remark  that  in  some  experiments  a  notable  oedema 
has  been  produced  in  guinea-pigs  at  the  point  of  inoculation,  while  in  others 
there  has  JDeen  no  local  lesion.  The  most  marked  oedema  resulted  from  cul- 
tures obtained  from  cases  of  measles. 

4 '  Do  the  facts  which  we  have  reported  explain  the  question  which  occupies 
us  ?  Can  we  conclude  that  there  is  a  relation  between  the  two  bacilli  ?  On 
the  one  side,  the  presence  of  the  pseudo  diphtheritic  bacillus  in  the  mouths  of 
healthy  persons,  and  of  those  who  have  anginas  manifestly  not  diphtheritic, 
seems  to  be  opposed  to  the  idea  of  a  relationship  between  them.  On  the 
other  hand,  when  we  consider  that  the  non- virulent  bacillus  is  very  rare  in 
fatal  diphtheria,  that  it  is  more  abundant  in  benign  diphtheria,  that  it  be- 
comes more  common  in  severe  cases  as  they  progress  towards  recovery,  and, 
finally,  that  they  are  more  numerous  in  persons  who  have  recently  had 
diphtheria  than  111  healthy  persons,  it  is  difficult  to  accept  the  idea  that  the 
two  microbes  are  entirely  distinct.  The  morphological  differences  which 
have  been  referred  to  are  so  slight  that  they  prove  nothing.  The  two  micro- 
organisms can  only  be  distinguished  by  their  action  upon  animals,  but  the 
difference  of  virulence  does  not  at  all  correspond  with  the  difference  of  ori- 
gin. As  regards  the  form  and  the  aspect  of  cultures,  the  true  and  false 
diphtheria  bacilli  differ  less  than  virulent  anthrax  differs  from  a  very  attenu- 
ated anthrax  bacillus,  which,  however,  originate  from  the  same  source. 
Besides,  the  sharp  distinction  which  we  make  between  the  virulent  and  non- 
virulent  bacilli  is  arbitrary;  it  depends  upon  the  susceptibility  of  guinea- 
pigs.  If  we  inoculate  animals  still  more  susceptible,  there  are  pseudo  diph- 
theritic bacilli  which  we  must  class  as  virulent;  and  if,  on  the  contrary,  we 
substitute  rabbits  for  guinea  pigs  in  our  experiments,  there  are  diphtheritic 
bacilli  which  we  must  call  pseudo-diphtheritic.  In  our  experiments  we  do 
not  simply  encounter  bacilli  which  are  very  virulent  and  bacilli  which  are 
non- virulent;  between  these  two  extremes  there  are  bacilli  of  every  degree 
of  virulence." 

Abbott,  in  1801,  published  the  result  of  his  researches  with 
reference  to  the  presence  of  the  pseudo-diphtheritic  bacillus  in 
benign  throat  affections.  He  made  a  bacteriological  study  of  fifty- 
three  patients,  nine  of  whom  were  suffering  from  acute  pharyngitis, 
fourteen  from  acute  follicular  tonsillitis,  eight  from  ordinary  post- 


382  BACTERIA   INT   DIPHTHERIA. 

nasal  catarrh,  two  from  simple  enlarged  tonsils,  fifteen  from  chronic 
pharyngitis,  one  from  subacute  laryngitis,  one  from  chronic  laryngi- 
tis, one  from  rhinitis,  and  two  from  an  affection  of  the  tonsils  and 
pharynx.  In  forty-nine  cases  nothing  of  particular  interest  was  ob- 
served. A  variety  of  microorganisms  were  isolated,  and  of  these 
the  pyogenic  micrococci  were  the  most  common. 

In  four  cases  microorganisms  were  found  which  resembled  the 
Bacillus  diphtheria  of  Loffler  in  their  morphology  and  growth  in  cul- 
ture media,  but  which  proved  not  to  be  pathogenic.  Abbott  says  : 
'  *  The  single  point  of  distinction  that  can  be  made  out  between  the 
organisms  obtained  from  Cases  L,  III.,  and  IV.  and  the  true  bacil- 
lus of  diphtheria  is  in  the  absence  of  pathogenic  properties  from  the 
former,  whereas  in  addition  to  this  point  of  distinction  the  organism 
from  Case  II.  gives,  as  has  been  stated,  a  decided  and  distinct 
growth  upon  the  surface  of  sterilized  potato. "  . 

40.    BACILLUS   DIPHTHERIA   COLUMBRARUM. 

Described  by  Loffler  (1884),  who  obtained  it  from  diphtheritic  pseudo-mem- 
branes in  the  mouths  of  pigeons  dead  from  an  infectious  form  of  .diphtheria 
which  prevails  in  some  parts  of  Germany  among  these  birds  and  among 
chickens. 

Reddened  patches  first  appear  upon  the  mucous  membrane  of  the  mouth 
and  fauces,  and  these  are  covered  later  with  a  rather  thick,  yellowish  layer 
of  fihrinous  exudaie.  In  pigeons  the  back  part  of  the  tongue,  the  fauces, 
and  tin*  corners  of  the  mouth  are  especially  affected;  in  chickens  the  tongue, 
the  £iiiiis,  the  nares,  the  larynx,  and  the  conjunct! val  mucous  membrane. 
The  disease  is  especially  fatal  among  chickens,  the  voung  fowls  and  those  of 
choice  varieties  oeing  most  susceptible.  It  is  attended  at  the  outset  by  fever, 
and  usually  proves  fatal  within  two  or  three  weeks,  but  may  last  for  several 
months. 

Morphology. — Short  bacilli  with  rounded  ends,  usually  associated  in  ir- 
regular masses,  and  resembling  the  bacilli  of  rabbit  septicaemia  (fowl 
cholera),  but  a  little  longer  and  not  quite  so  broad.  In  sections  from  the 
liver  they  are  seen  in  irregular  groups  in  the  interior  of  the  vessels. 

Biological  Characters. — An  aerobic,  non-motile,  non-liquefying  bacillus. 
(i rows  in  nutrient  gelatin  in  the  form  of  spherical,  white  colonies  along 
the  line  of  puncture,  and  upon  the  surface  as  a  whitish  layer.  Under  the 
microscope  the  colonies  in  gelatin  plates  have  a  yellowish-brown  color  and 
a  slightly  granular  surface.  Upon  blood  serum  the  growth  consists  of  a 
-•  mi-transparent,  grayish-white  layer.  Upon  potato  a  thin  layer  is  formed 
1  laving  a  grayish  tint. 

ruthogenesift. — Pigeons  inoculated  with  a  pure  culture  in  the  mucous 
membrane  of  the  mouth  are  affected  exactly  as  are  those  which  acquire  the 
naturally.  Subcutaneous  inoculations  in  pigeons  ^ive  rise  to  an  in- 
tlammation  resulting  in  local  necrotic  changes.  Pathogenic  for  rabbits  and 
for  mice.  Suhcutaneous  injections  in  mice  give  rise  toa  fatal  result  in  about 
live  <lays.  The  bacillus  is  found  in  the  blood  and  in  the  various  organs,  in 
the  interior  of  the  vessels,  and  sometimes  in  the  interior  of  the  leucocytes; 
they  are  especially  numerous  in  the  liver.  The  lungs  are  dotted  with  red 
spots,  the  spleen  is  greatly  enlarged,  and  the  liver  has  a  marbled  appearance 
from  the  presence  of  numerous  irregular  white  masses  scattered  through  the 
p.-ile-red  parenchyma  of  the  organ.  These  white  masses  are  seen,  in  sec- 
tions, to  consist  of  necrotic  liver  tissue,  iu  the  centre  of  which  the  bacilli 


BACTERIA   IN   DIPHTHERIA.  383 

are  found  in  great  numbers,  in  the  interior  of  the  vessels.  This  appearance 
is  so  characteristic  that  Loffler  considers  inoculations  in  mice  to  be  the  most 
reliable  method  of  establishing  the  identity  of  the  bacillus.  Not  pathogenic 
for  chickens,  guinea-pigs,  rats,  or  dogs. 

There  seems  to  be  some  doubt  whether  the  form  of  diphtheria  which  pre- 
vails among  pigeons,  and  which  Loffler  has  shown  to  be  due  to  the  bacillus 
above  described,  is  identical  with  the  diphtheria  of  chickens.  Diphtheria  in 
man  has  been  supposed  by  some  authors  to  be  identical  with  that  which 
prevails  among  fowls,  and  possibly  this  may  be  the  case  under  certain  cir- 
cumstances. But  the  evidence  seems  to  be  convincing  that  there  is  an 
infectious  diphtheria  of  fowls  which  is  peculiar  to  them,  and  which,  under 
ordinary  circumstances,  is  not  communicated  to  man. 

50.    BACILLUS   DIPHTHERIA  VITULORUM. 

Described  by  Loffler  (1884)  and  obtained  by  him  from  the  pseudo-mem- 
branous exudation  in  the  mouths  of  calves  suffering  from  an  infectious  form 
of  diphtheria.  The  disease  is  characterized  by  the  appearance  of  yellow 
patches  upon  the  mucous  membrane  of  the  cheeks,  the  gums,  the  tongue, 
and  sometimes  of  the  larynx  and  nares  of  infected  animals.  There  is  a  yel- 
lowish discharge  from  the  nose,  an  abundant  flow  of  saliva,  occasional  at- 
tacks of  coughing,  and  diarrhoea.  Death  may  occur  at  the  end  of  four  or 
five  days,  but  usually  the  animal  survives  for  several  weeks.  Diphtheritic 
patches  similar  to  those  in  the  mouth  are  also  found  in  the  large  intestine, 
and  scattered  abscesses  in  the  lungs. 

Loffler,  in  a  series  of  seven  cases  examined,  obtained  from  the  deeper  por- 
tions of  the  pseudo-membranous  deposit  a  long  bacillus  which  appears  to  be 
the  cause  of  the  disease. 

Morphology. — Bacilli,  five  to  six  times  as  long  as  broad,  usually  united  in 
long  filaments.  The  diameter  of  the  rods  is  about  half  that  of  the  bacillus 
of  malignant  oedema. 

Biological  Characters.—  Attempts  to  cultivate  this  bacillus  in  nutrient 
gelatin,  blood  serum  from  sheep,  and  various  other  media  were  unsuccessful. 
But  when  fragments  of  tissue  containing  the  bacillus  were  placed  in  blood 
serum  from  the  calf  a  whitish  border,  consisting  of  the  long  bacilli,  was  de- 
veloped. These  could  not,  however,  be  made  to  grow  when  transferred  to 
fresh  blood  serum. 

Pathogenesis. —  Mice  inoculated  subcutaneously  with  the  fresh  diph- 
theritic exudation  died  in  from  seven  to  thirty  days.  The  autopsy  disclosed 
an  extensive  infiltration  of  the  entire  walls  of  the  abdomen,  which  often  pene- 
trated the  peritoneal  cavity  and  enveloped  the  liver,  the  kidneys,  and  the 
intestine  in  a  yellowish  exudate.  The  bacillus  was  found  in  this  exudate, 
and  by  inoculating  a  little  of  it  into  another  animal  of  the  same  species  a 
similar  result  was  obtained.  Not  pathogenic  for  rabbits  or  guinea-pigs. 

51.    BACILLUS   OF   INTESTINAL   DIPHTHERIA   IN  RABBITS. 

Described  by  Ribbert  (1887)  and  obtained  by  him  from  the  organs  of  rab- 
bits which  succumbed  to  an  affection  characterized  by  a  diphtheritic  inflam- 
mation of  the  mucous  membrane  of  the  intestine.  The  autopsy  revealed  also 
swelling  of  the  mesenteric  glands  and  minute  iiecrotic  foci  in  the  liver  and 
spleen. 

Morphology. — Bacilli  with  slightly  rounded  ends,  from  three  to  four/* 
long  and  1  to  1.4  //  in  diameter;  often  united  in  pairs  or  in  filaments  con- 
taining several  elements. 

Stains  with  the  aniline  colors,  but  not  so  readily  in  sections  as  some 
other  microorganisms.  Ribbert  recommends  staining  with  aniline-water- 
fuchsin  solution,  washing  in  water,  then  placing  the  sections  in  methylene 
blue  solution,  and  decolorizing  in  alcohol.  Does  not  stain  by  Gram's 
method. 


BACTERIA   IN  DIPHTHERIA. 

Biological  Characters.— An  aerobic,  non-liquefying  (non -motile  ?)  ba- 
cillus. Upon  gelatin  plates  semi-transparent,  grayish  colonies  are  formed, 
which  later  have  a  brownish  color;  the  surface  of  these  is  finely  granular 
and  of  a  pearly  lustre.  In  stick  cultures  in  nutrient  gelatin  the  growth 
along  the  line  of  puncture  is  very  scanty.  On  potato  a  flat,  whitish  layer  is 
formed,  which  extends  slowly  over  the  surface.  Grows  best  at  a  temperature 
of  ;;u  t..:{;V  C. 

Pathogenesis.—PurQ  cultures  injected  into  the  peritoneal  cavity  or  sub- 
cutaneously  in  rabbits  caused  the  death  of  these  animals  in  from  three  to 
fourteen  days,  according  to  the  quantity  injected.  At  the  autopsy  necrotic 
foci  are  found  in  the  liver  and  spleen,  and  the  mesenteric  glands  are  en- 
larged, but  the  intestine  presents  a  healthy  appearance.  But  when  cultures 
are  introduced  into  the  alimentary  canal  the  characteristic  diphtheritic  in- 
flammation of  the  mucous  membrane  of  the  intestine  is  induced.  This  re- 
sult was  obtained  both  by  direct  injection  into  the  lumen  of  the  intestine 
and  by  injecting  cultures  into  the  mouth. 

Additional  Notes  upon  Diphtheria  and  the  Diphtheria  Bacil- 
lus.— C.  Frankel  (1895)  reports  that  he  has  repeatedly  observed 
branching  forms  of  the  diphtheria  bacillus  in  cultures  upon  Lof- 
fler's  blood-serum  medium,  and  that  these  branching  forms  are  seen 
more  constantly  and  in  greater  numbers  in  cultures  made  upon  the 
surface  of  hard-cooked  albumen  from  hen's  eggs. 

The  continued  presence  of  virulent  diphtheria  bacilli  in  the  fauces 
of  patients  who  have  recovered  from  the  disease,  either  after  the  use 
of  the  antitoxin  or  under  other  treatment,  has  been  demonstrated  by 
several  bacteriologists.  Silverschmidt  (1895),  in  forty-five  cases 
treated  by  Behring's  antitoxic  serum,  found  that  the  number  of  ba- 
cilli usually  diminished  some  days  after  the  treatment  was  com- 
menced, but  that  in  cases  in  which  complete  recovery  had  taken 
place  not  infrequently  virulent  bacilli  could  be  obtained  many  days 
(in  one  case  thirty-one  days)  after  convalescence  was  established. 

Escherich  (1893)  opposes  the  view  that  the  pseudo-diphtheria  bacil- 
lus is  simply  a  non-virulent  variety  of  the  diphtheria  bacillus.  He 
found  this  pseudo-diphtheria  bacillus  in  the  throats  of  thirteen  out 
of  three  hundred  and  twenty  individuals  examined.  According  to 
lii in  there  is  no  evidence  that  this  completely  non-virulent  pseudo- 
diphtheria  bacillus  ever  acquires  pathogenic  virulence,  while  attenu- 
ated varieties  of  the  true  diphtheria  bacillus  readily  recover  their 
power  to  produce  the  toxic  products  upon  which  virulence  depends. 

Sevestre  (1895),  as  a  result  of  researches  made  by  himself  and 
several  other  bacteriologists  who  have  made  similar  investigations, 
arri  ves  at  the  conclusion  that : 

"First.  In  a  <vrtain  number  of  cases  the  bacillus  of  Loffler  disap- 
pears about  the  same  time  as  the  false  membranes;  or  it  may  persist 
t'<>r  sum.-  time,  but  ceases  to  be  virulent— in  this  case  it  seems  to  have 
undergone  modifications  and  presents  tbe  form  of  short  bacilli. 

"  Second.  In  another  series  of  cases,  less  numerous  but  neverthe- 


BACTERIA   IN   DIPHTHERIA.  385 

less  considerable,  the  bacillus  persists  in  a  virulent  condition  for  a 
longer  or  shorter  time  after  the  apparent  cure  of  the  malady.  .  . 

"  Third.  The  observations  collected  up  to  the  present  time  do  not 
enable  us  to  fix  precisely  the  limits  of  persistence,  but  it  is  not  far 
out  of  the  way  if  we  place  it  at  several  weeks  to  a  month  for  the 
throat.  In  the  nasal  fossa3  the  bacillus  often  persists  for  a  still 
longer  time,  and  its  presence  commonly  coincides  with  a  more  or  less 
abundant  discharge  from  the  nose." 

Park  and  Beebe  (1894),  in  an  extended  research  made  for  the  pur- 
pose of  determining  the  persistence  of  the  diphtheria  bacillus  in  the 
throats  of  convalescents  (2,560  cultures  made),  found  that  in  304  out 
of  605  consecutive  cases  the  bacillus  disappeared  within  3  days  after 
the  disappearance  of  the  exudate;  in  176  cases  it  persisted  for  7  days; 
in  64  cases  for  12  days;  in  36  cases  for  15  days;  in  12  cases  for  3 
weeks;  in  4  cases  for  4  weeks;  in  2  cases  for  9  weeks.  Park  and 
Beebe  arrive  at  the  following  conclusion  with  reference  to  pseudo- 
diphtheria  bacilli : 

"  The  name  pseudo-diphtheria  bacillus  should  be  regarded  as  ap- 
plying to  those  bacilli  found  in  the  throat  which,  though  resembling 
the  diphtheria  bacilli  in  many  respects,  yet  differ  in  others  equally  im- 
portant. These  bacilli  are  rather  short,  and  more  uniform  in  size 
and  shape  than  the  typical  Loffler  bacillus.  They  stain  equally 
throughout  with  the  alkaline  methyl-blue  solution,  and  produce 
alkali  in  their  growths  in  bouillon.  They  are  found  in  about  one 
per  cent  of  the  healthy  throats  in  New  York  City,  and  seem  to  have 
no  connection  with  diphtheria.  They  are  never  virulent." 

Park  (1894)  has  shown  that  virulent  diphtheria  bacilli  are  fre- 
quently found  in  the  throats  of  persons  who  have  been  associated 
with  diphtheria  patients,  although  no  manifestations  of  the  disease 
were  visible.  It  is  therefore  apparent  that  infection  requires  not 
only  the  presence  of  virulent  bacilli,  but  also  of  a  predisposition  to 
the  disease.  This  corresponds  with  the  facts  relating  to  other  in- 
fectious diseases — e.g.^  tuberculosis,  typhoid  fever — and  among  the 
probable  predisposing  causes  we  may  mention  "  sewer-gas  poisoning, " 
catarrhal  inflammations  of  the  mucous  membranes  most  commonly 
involved,  inanition,  "crowd  poisoning,"  and  depressing  agencies 
generally. 

Bacteriologists  have  recently  given  much  attention  to  the  question 
of  mixed  infection  in  diphtheria.  Funck  (1894)  accepts  the  gener- 
ally received  view  that  mixed  infections  with  the  diphtheria  bacillus 
and  Streptococcus  pyogenes  are  more  serious  than  an  uncomplicated 
diphtheria,  and  in  an  experimental  research  has  attempted  to  deter- 
mine whether  this  is  due  to  an  increased  production  of  the  diphtheria 
the  presence  of  the  streptococcus.  His  experiments  on  guinea-pigs 
27 


386  BACTERIA   IN   DIPHTHERIA. 

showed  that  when  infected  with  streptococci  these  animals  did  not 
prove  to  be  more  sensitive  to  the  action  of  the  diphtheria  poison 
(without  living  bacilli),  and  he  concludes  that  the  unfavorable  influ- 
ence of  the  streptococcus  in  mixed  infections  is  due  to  increased  patho- 
genic activity  on  the  part  of  the  diphtheria  bacillus.  Bernheim 
(1894)  found,  in  his  experiments  on  guinea-pigs,  that  they  suc- 
cumbed more  rapidly  to  diphtheria  infection  when  they  previously 
or  simultaneously  received  an  injection  of  a  streptococcus  culture- 
filtered  or  unfiltered. 

Results  of  Treatment  ivith  the  Antitoxin. — While  questions  re- 
lating to  therapeutics  are  not  considered  in  this  manual,  a  brief  note 
upon  the  results  of  treatment  by  the  serum  of  immunized  animals 
may  not  be  out  of  place.  A  recent  (1895)  collective  investigation 
undertaken  by  the  Deutsche  medicinische  Wochenschrift  gave  the 
following  results :  The  number  of  cases  collected  was  10,312;  all  of 
these  occurred  between  the  1st  of  October,  1894,  and  the  1st  of  April, 
1895;  5,883  of  these  cases  were  treated  with  the  antitoxin  and  4,479 
without  it.  In  the  first  group  the  mortality  was  9.6  per  cent,  and  in 
the  second  group  14.7  per  cent.  Two  thousand  five  hundred  and  fifty 
six  children  treated  with  the  antitoxin  were  between  two  and  ten 
years  of  age;  among  these  the  mortality  was  4  per  cent,  while 
among  children  of  the  same  age  not  treated  with  the  antitoxin  the 
mortality  was  15.2  per  cent.  Six  hundred  and  ninety-six  patients 
above  ten  years  of  age  were  treated  with  a  mortality  of  1  per  cent. 

Monod  (1895),  at  a  meeting  of  the  Paris  Academy  of  Medicine, 
presented  the  following  statistics  demonstrating  the  influence  upon 
the  mortality  from  diphtheria  in  France  exerted  by  the  antitoxin 
since  its  employment  from  November,  1894.  The  following  figures 
represent  the  number  of  deaths  from  diphtheria  during  the  first  six 
months  in  eight  years  in  108  French  cities  having  a  population  of 
more  than  20,000: 


1805, 

Average.         Average. 

January 469  205 

February 466  187 

March 499  155 

April 442  160 

.     May 417  113 

June 333  84 

2,656  904 

It  will  be  seen  from  the  above  statement  that  during  the  first  six 
months  in  the  year  1895  after  the  introduction  of  the  antitoxin  treat- 
ment, the  number  of  deaths  from  diphtheria  in  the  108  French  cities 
referred  to  was  1,552  less  than  the  average  for  the  preceding  ten 
years,  and  we  are  justified  in  concluding  that  a  considerable  propor- 
tion of  this  saving  at  least  is  due  to  this  new  method  of  treatment. 


X. 
BACTERIA   IN  INFLUENZA. 

A  NUMBER  of  bacteriologists  have  made  careful  researches  during 
the  recent  extended  epidemic  of  influenza,  and  in  1892  a  bacillus 
was  discovered,  both  by  Pfeiffer  and  by  Canon,  of  Berlin,  which 
there  is  good  reason  to  believe  is  the  specific  cause  of  this  dis- 
ease. Before  describing  this  we  shall  refer  briefly  to  previous  re- 
searches. 

Babes  has  described  no  less  than  seventeen  distinct  species  or  varieties 
isolated  by  him,  principally  from  nasal  or  bronchial  mucus.  Among  these 
a  considerable  number  closely  resemble  Streptococcus  pyogenes  or  Micro - 
coccus  pneumonias  crouposae.  No  one  form  was  found  with  sufficient  con- 
stancy to  justify  the  inference  that  it  was  the  specific  cause  of  the  disease. 

Klebs,  in  examining  blood  drawn  from  the  fingers  of  patients  with  influ- 
enza, observed  an  enormous  number  of  small,  actively  motile,  highly  refrac- 
tive bodies,  which  in  their  size,  form,  and  movements  corresponded  entirely 
with  similar  bodies  previously  observed  by  him  in  the  blood  of  patients  with 
pernicious  anaemia,  but  whicn  were  far  more  numerous.  These  bodies  are 
believed  by  Klebs  to  be  flagellate  infusoria  ("flagellata").  The  investiga- 
tions of  other  bacteriologists  have  not  thus  far  confirmed  those  of  Klebs  as 
regards  the  presence  of  microorganisms  of  this  class  in  the  blood  of  patients 
with  influenza. 

Kowalski,  who  made  bacteriological  researches  in  sixteen  cases,  was  not 
able  to  find  microorganisms  of  any  kind  in  the  blood,  examined  both  fresh 
and  in  dried  preparations.  In  his  cultures  from  the  nasal,  buccal,  and 
bronchial  secretions  of  the  sick  he  obtained  in  five  cases  Staphylococcus 
pyogenes  aureus,  in  four  Staphylococcus  pyogenes  albus,  in  two  "diplococ- 
cus  pneumonise, "  in  two  Streptococcus  pyogenes,  in  two  Staphylococcus 
pyogenes  citreus,  in  one  Friedlander's  bacillus,  in  one  Staphylococcus  cereus 
albus,  in  one  Staphylococcus  cereus  flavus.  In  addition  to  these  he  isolated 
three  species  not  previously  described.  One  of  these  he  found  in  seven 
cases  ;  this  grew  upon  the  surface  of  agar  as  small,  transparent  drops,  but 
did  not  grow  upon  potato,  in  sterilized  milk,  or  in  bouillon ;  it  was  a  coccus 
arranged  in  pairs  or  in  chains,  and  is  designated  by  Kowalski  ' '  Gallertstrep- 
tococcus." 

Prior,  in  a  bacteriological  study  of  fifty- three  cases,  twenty-nine  of  which 
were  without  complication  and  twenty-four  complicated  by  pneumonia, 
found  in  the  sputum  of  uncomplicated  cases,  as  the  most  abundant  and  com- 
mon microorganism  at  the  outset  of  the  attack,  Micrococcus  pneumonias 
crouposse ;  next  to  this  came  Staphylococcus  pyogenes  aureus  and  Strepto- 
coccus pyogenes ;  when  the  acme  of  the  attack  was  past  the  two  species  first 
named  quickly  diminished  in  numbers,  while  streptococci  were  found  for  a 
longer  time.  In  cases  of  croupous  pneumonia  following  influenza  "  diplo- 
coccus  pneumonias  "  was  constantly  found  in  great  numbers. 

Fischel  (1891)  obtained  in  cultures  from  the  blood  of  two  cases  two  dif- 


388  BACTERIA  IN  INFLUENZA. 

ferentmicrococci,  one  of  which  was  pathogenic  for  dogs  and  horses  and  gave 
rise  to  symptoms  in  these  animals  resembling  those  of  influenza  (see  Micro- 
coccus  No.  II.  of  Fischel,  No.  39,  nage  324). 

Kirchner  (1891)  found  constantly  in  the  sputum  of  recent  cases  a  diplo- 
coccus  enclosed  in  a  jellv-like  capsule,  which  differed  in  its  biological  and 
pathological  characters  from  Micrococcus  pneumonioe  crouposae  (see  Micro- 
coccus  of  Kirchner,  No.  38,  page  324). 

52.    BACILLUS  OF  INFLUENZA. 

Discovered  by  Pfeiffer  (1892)  in  the  purulent  bronchial  secretion, 
and  by  Canon  in  the  blood  of  patients  suffering  from  epidemic  in- 
fluenza. Pfeiffer  found  the  bacillus  in  thirty-one  cases  examined  by 
him,  and  in  uncomplicated  cases  it  was  present  in  the  purulent  bron- 
chial secretion  in  immense  numbers  and  in  a  pure  culture.  Canon, 
whose  independent  observations  were  published  at  the  same  time, 
examined  the  blood  of  twenty  influenza  patients  in  stained  prepara- 
tions, and  found  the  same  bacillus  in  nearly  all  of  them.  His  method 
of  demonstrating  it  is  as  follows  : 

The  blood  is  spread  upon  clean  glass  covers  in  the  usual  way. 
After  the  preparations  are  thoroughly  dry  they  are  placed  in  abso- 
lute alcohol  for  five  minutes.  They  are  then  transferred  to  the  fol- 
lowing staining  solution  (Czenzynke's) :  concentrated  aqueous  solu- 
tion of  methylene  blue,  forty  grammes  ;  one-half -per-cent  solution  of 
eosin  (dissolved  in  seventy-per-cent  alcohol),  twenty  grammes  ;  dis- 
tilled water,  forty  grammes.  The  cover  glasses  immersed  in  this 
staining  solution  are  placed  in  an  incubating  oven  at  37°  C.  for  from 
three  to  six  hours,  after  which  they  are  washed  with  water,  dried, 
and  mounted  in  balsam.  In  successful  preparations  the  red  blood 
corpuscles  are  stained  red  by  the  eosin,  and  the  leucocytes  blue.  The 
bacillus  is  seen  in  these  as  a  short  rod,  often  resembling  a  diplococcus. 
It  is  sometimes  seen  in  large  numbers,  but  usually  only  a  few  rods 
are  seen  after  a  long  search — four  to  twenty  in  a  single  preparation. 
In  six  cases  it  was  found  in  numerous  aggregations  containing  from 
five  to  fifty  bacilli  each.  In  these  cases  the  blood  was  drawn  during 
a  fall  of  temperature  or  shortly  after. 

Morphology.— Very  small  bacilli,  having  about  the  same  diameter 
as  the  bacillus  of  mouse  septicaemia,  but  only  half  as  long.  Solitary 
or  united  in  chains  of  three  or  four  elements. 

Stains  with  difficulty  with  the  basic  aniline  dyes— best  with 
dilute  Ziehl's  solution,  or  Loffler's  methylene  blue  solution,  with  heat. 
The  two  ends  of  the  bacilli  are  most  deeply  stained,  causing  them  to 
resemble  diplococci.  Pfeiffer  says:  "I  am  inclined  to  believe  that 
some  of  the  earlier  observers  also  saw  the  bacilli  described  by  me, 
but  that,  misled  by  their  peculiar  behavior  with  regard  to  staining 
agents,  they  described  them  as  diplococci  or  streptococci."  Do  not 
stain  hy  (iram's  method. 


BACTERIA   IN   INFLUENZA.  389 

Biological  Characters. — An  aerobic,  non-motile  bacillus.  Does 
not  grow  in  nutrient  gelatin  at  the  room  temperature.  Spore  forma- 
tion not  observed .  Upon  the  surface  of  glycerin-agar  in  the  incubat- 
ing oven  very  small,  transparent,  drop-like  colonies  are  developed  at 
the  end  of  twenty-four  hours.  These  can  only  be  recognized  by  the 
aid  of  a  lens.  "  A  remarkable  point  about  them  is  that  the  colonies 
always  remain  separate  from  each  other,  and  do  not,  as  all  other 
species  known  to  me  do,  join  together  and  form  a  continuous  row. 
This  feature  is  so  characteristic  that  the  influenza  bacilli  can  be 
thereby  with  certainty  distinguished  from  other  bacteria"  (Kitasato). 
On  1.5  per  cent  sugar-agar  the  colonies  appear  as  extremely  small 
droplets,  clear  as  water,  often  only  recognizable  with  a  lens 
(Pfeiffer). 

In  bouillon  a  scanty  development  occurs,  and  at  the  end  of  twen- 
ty-four hours  small,  white  particles  are  seen  upon  the  surface,  which 
subsequently  sink  to  the  bottom,  forming  a  white,  woolly  deposit, 
while  the  bouillon  above  remains  transparent.  This  bacillus  does 
not  grow  at  temperatures  below  28°  C. 

Canon  has  obtained  colonies,  resembling  those  described  by  Kita- 
sato, in  cultures  from  the  blood  of  influenza  patients.  His  cultures 
were  made  upon  glycerin-agar  in  Petri's  dishes.  Ten  or  twelve  drops 
of  blood  from  a  puncture  made  in  the  finger  of  the  patient,  after 
sterilization  of  the  surface,  were  allowed  to  fall  upon  the  agar  medium, 
and  this  was  placed  in  the  incubating  oven.  As  the  number  of  ba- 
cilli in  the  blood  is  small,  a  considerable  quantity  is  used.  The 
colonies  are  visible  at  the  end  of  twenty-four  to  forty-eight  hours. 

The  influenza  bacillus  is  quickly  destroyed  by  desiccation  ;  a 
pure  culture  diluted  with  water  and  dried  is  destroyed  with  cer- 
tainty in  twenty  hours ;  in  dried  sputum  the  vitality  is  retained 
somewhat  longer,  but  no  growth  occurs  after  forty  hours.  The 
thermal  death-point  is  60°  C.  with  five  minutes'  exposure  (Pfeiffer 
and  Beck). 

Patliogenesis. — Pfeiffer  infers  that  this  is  the  specific  cause  of 
influenza  in  man  for  the  following  reasons  : 

1.  They  were  found  in  all  uncomplicated  cases  of  influenza  ex- 
amined, in  the  characteristic  purulent  bronchial  secretion,  often  in 
absolutely  pure  cultures.     They  were  frequently  situated  in  the  pro- 
toplasm of  the  pus  corpuscles  ;   in  fatal  cases  they  were  found  to 
have  penetrated  from  the  bronchial  tubes  into  the  peribronchitic  tis- 
sue, and  even  to  the  surface  of  the  pleura,  where  in  two  cases  they 
were  found  in  pure  cultures  in  the  purulent  exudation. 

2.  They  were  only  found  in  cases  of  influenza.     Numerous  con- 
trol experiments  proved   their  absence   in   ordinary  bronchial  ca 
tarrh,  etc. 


390  BACTERIA   IN   INFLUENZA. 

3.  The  presence  of  the  bacilli  corresponded  with  the  course  of  the 
disease,  and  they  disappeared  with  the  cessation  of  the  purulent 
bronchial  secretion. 

In  his  preliminary  report  of  his  investigations  Pfeiffer  says  : 

"  Numerous  inoculation  experiments  were  made  on  apes,  rabbits, 
guinea-pigs,  rats,  pigeons,  and  mice.  Only  in  apes  and  rabbits 
could  positive  results  be  obtained.  The  other  species  of  animals 
showed  themselves  refractory  to  influenza." 

Kruse  (1894)  reports  that  he  found  the  bacillus  of  Pfeiffer  in 
eighteen  influenza  patients  examined  by  him  in  the  hospital  at  Bonn. 
On  the  other  hand,  he  failed  to  find  it  in  a  considerable  number  of  pa- 
tients suffering  from  other  diseases  of  the  respiratory  passages.  His 
evidence  is  the  more  valuable  as  he  had  previously  (1890)  reported 
his  failure  to  find  the  bacillus  in  typical  cases  of  influenza.  He  now 
ascribes  his  failure  at  that  time  to  imperfect  technique. 

Huber  (1893),  Richter  (1894),  Borchardt  (1894),  and  other  com- 
petent bacteriologists,  have  also  confirmed  the  results  reported  by 
Pfeiffer  as  regards  the  presence  of  this  bacillus  in  the  bronchial 
secretions  of  persons  suffering  from  epidemic  influenza,  and  as  to 
its  biological  characters.  Bujwid  (1893)  recognizes  the  bacillus  of 
Pfeiffer  as  identical  with  a  bacillus  which  he  cultivated  from  the 
spleen  of  an  influenza  patient  in  1890. 

The  researches  of  Weichselbaum,  Kowalski,  Friedrich,  Kruse, 
Bouchard,  and  others  have  given  a  negative  result  as  regards  the 
presence  of  the  influenza  bacillus  in  the  blood.  They  were  not  able 
to  demonstrate  its  presence  either  in  stained  preparations  or  by  cul- 
ture methods.  Pfeiffer,  also,  during  the  last  epidemic,  has  made 
special  researches  upon  this  point  and  has  never  succeeded  in  finding 
the  bacillus.  Day  after  day,  both  in  mild  and  severe  cases,  he  placed 
from  ten  to  twenty  drops  of  blood  from  influenza  patients  on  blood- 
agar — a  most  favorable  medium — but  his  cultures  always  remained 
sterile. 

In  his  experiments  upon  rabbits,  Pfeiffer  (1893)  found  that  the 
intravenous  injection  of  a  small  quantity  of  culture  on  blood-agar, 
twenty-four  hours  old,  suspended  in  one  cubic  centimetre  of  bouillon, 
caused  a  characteristic  pathogenic  effect.  The  first  symptoms  were 
developed  within  one  and  a  half  to  two  hours  after  the  injection. 
The  animals  became  extremely  feeble,  lying  flat  upon  the  floor  with 
their  limbs  extended,  and  suffered  from  extreme  dyspnoea.  The  tem- 
perature mounted  to  41°  C.  or  above.  At  the  end  of  five  or  six  hours 
they  were  able  to  sit  upon  their  haunches  again,  and  in  twenty-four 
hours  had  nearly  recovered  from  all  indications  of  ill-health.  Larger 
doses  caused  the  death  of  the  inoculated  animals.  These  results  are 
due  to  toxic  products  present  in  the  cultures,  and  Pfeiffer  has  never 


PLATE  VI. 

PATHOGENIC  BACTERIA. 

FIG.  1.— Bacillus  of  influenza  in  bronchial  mucus.  X  1,000.  Photo- 
micrograph by  Frankel. 

FIG.  2.— Bacillus  of  influenza  in  bronchial  mucus,  after  the  termination 
of  the  febrile  period.  The  bacilli  are  for  the  most  part  in  pus  cells.  X  1,000. 
Photomicrograph  by  Frankel. 

FIG.  «3. — Bacillus  tetani  from  an  agar  culture.  X  1,000.  Photomicro- 
graph by  Frankel  and  Pfeiffer. 

FIG.  4. — Micrococcus  pneumoniae  crouposae  in  sputum  of  a  patient  with 
pneumonia.  X  1,000.  Stained  by  Gram's  method.  Photomicrograph  by 
Frankel  and  Pfeiffer. 

FIG.  5. — Micrococcus  pneumoniae  crouposae  in  blood  of  rabbit.  X  1,000. 
Photomicrograph  made  at  the  Army  Medical  Museum,  Washington,  by  Gray. 

FIG.  6.— Bacillus  of  hog  cholera,  showing  flagella.  Stained  by  Loffler's 
method.  X  1,000.  Photomicrograph  made  at  the  Army  Medical  Museum, 
Washington,  by  Gray. 


TLAlh   VI. 

STERNBERG'S  BACTERIOLOGY.' 


Fig.     3. 


Fie:.    4. 


Fig.     5, 


'""••.•>:• 


Fig.    0. 


PATHOGENIC  BACTERIA. 


BACTERIA  IN   INFLUENZA.  391 

observed  a  septicsemic  infection  as  a  result  of  his  inoculation  ex- 
periments. 

Pfeiffer  has  found  in  three  cases  of  bronchopneumonia  a  pseudo- 
influenza  bacillus  which  closely  resembles  the  bacillus  previously  de- 
scribed by  him  as  peculiar  to  that  disease.  This  pseudo-influenza 
bacillus  resembles  the  genuine  one  in  its  growth  in  culture  media, 
but  is  larger  and  shows  a  decided  inclination  to  grow  out  into  long 
threads.  By  these  morphological  characters,  which  are  said  to  be 
constant,  it  may,  according  to  Pfeiffer,  be  readily  distinguished. 


XL 
BACILLI  IN  CHRONIC  INFECTIOUS  DISEASES. 

IN  tuberculosis,  leprosy,  glanders,  and  syphilis  we  have  a  group 
of  infectious  diseases  which  present  many  points  of  resemblance. 
All  run  a  chronic  course  ;  all  may  be  communicated  to  susceptible 
animals  by  inoculation  ;  in  all,  the  lymphatic  glands  in  the  vicinity 
of  the  point  of  inoculation  become  enlarged,  and  new  growths,  con- 
sisting of  various  cellular  elements  of  a  low  grade  of  vitality,  are  de- 
veloped in  the  tissues  which  are  the  point  of  predilection  for  each  ; 
in  all,  these  new  growths  show  a  tendency  to  degenerative  changes, 
as  a  result  of  which  abscesses,  caseous  masses,  or  open  ulcers  are 
formed 

In  two  of  the  diseases  in  this  group — tuberculosis  and  glan- 
ders— the  infectious  agent  has  been  obtained  in  pure  cultures  and  its 
specific  pathogenic  power  demonstrated  by  inoculations  in  susceptible 
animals;  in  one — leprosy — there  is  but  little  doubt  that  the  bacillus  con- 
stantly found  in  the  new  growths  characteristic  of  the  disease  bears 
an  etiological  relation  to  it,  although  this  has  not  been  demonstrated, 
the  bacillus  not  having  as  yet  been  cultivated  in  artificial  media. 
The  evidence  with  reference  to  the  parasitic  nature  of  the  fourth  dis- 
ease mentioned  as  belonging  to  this  group — syphilis — is  still  unsatis- 
t.K-tory,  but  there  is  every  reason  to  believe  that  it  will  also  eventu- 
ally be,  proved  to  be  due  to  a  parasitic  microorganism. 

The  announcement  of  the  discovery  of  the  tubercle  bacillus  was 
made  by  Koch,  in  March,  1882,  at  a  meeting  of  the  Physiological 
S.  K-ioty  of  Berlin.  At  the  same  time  satisfactory  experimental  evi- 
dence was  presented  as  to  its  etiological  relation  to  tuberculosis  in 
man  and  in  the  susceptible  lower  animals,  and  its  principal  biologi- 
cal i-liaracters  were  given. 

Baumgarten  independently  demonstrated  the  presence  of  the  tu- 
bercle bacillus  in  tuberculous  tissues  and  published  the  fact  soon 
at't.T  tlu>  appearance  of  Koch's  first  paper.  The  previous  demonstra- 
tion by  VilK'inin  (lsr,r>)— confirmed  by  Cohnheim  (1877)  and  others— 
that  tuberculosis  might  be  induced  in  healthy  animals  by  inocula- 
tions of  tuberculous  material,  had  paved  the  way  for  his  discovery, 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

and  advanced  pathologists  were  quite  prepared  to  accept  it.  The 
more  conservative  have  since  been  obliged  to  yield  to  the  experi- 
mental evidence,  which  has  received  confirmation  in  all  parts  of  the 
world.  To-day  it  is  generally  recognized  that  tuberculosis  is  a  spe- 
cific infectious  disease  due  to  the  tubercle  bacillus. 

As  evidence  of  the  thorough  nature  of  Koch's  personal  researches 
in  advance  of  his  first  public  announcement,  we  give  the  following 
resume  of  his  investigations  : 

In  nineteen  cases  of  miliary  tuberculosis  the  bacilli  were  found  in 
the  tubercular  nodules  in  every  instance  ;  also  in  twenty-nine  cases 
of  pulmonary  phthisis,  in  the  sputum,  in  fresh  cheesy  masses,  and  in 
the  interior  of  recently  formed  cavities ;  in  tuberculous  ulcers  of  the 
tongue,  tuberculosis  of  the  uterus,  testicles,  etc.  ;  in  twenty-one  cases 
of  tuberculous — scrofulous — lymphatic  glands ;  in  thirteen  cases  of 
tuberculous  joints  ;  in  ten  cases  of  tubercular  bone  affections  ;  in  four 
cases  of  lupus  ;  in  seventeen  cases  of  Perlsucht  in  cattle.  His  ex- 
perimental inoculations  were  made  upon  two  hundred  and  seventy- 
three  guinea-pigs,  one  hundred  and  five  rabbits,  forty-four  field 
mice,  twenty-eight  white  mice,  nineteen  rats,  thirteen  cats,  and  upon 
dogs,  pigeons,  chickens,  etc.  Very  extensive  comparative  researches 
were  also  made,  which  convinced  him  that  the  bacillus  which  he  had 
been  able  to  demonstrate  in  tuberculous  sputum  and  tissues  by  a  spe- 
cial mode  of  staining  was  not  to  be  found  in  the  sputa  of  healthy 
persons,  or  of  those  suffering  from  non-tubercular  pulmonary  affec- 
tions, or  in  organs  and  tissues  involved  in  morbid  processes  of  a 
different  nature. 

53.    BACILLUS   TUBERCULOSIS. 

Discovered  by  Koch  (first  public  announcement  of  discovery 
March  24th,  1882).  The  bacilli  are  found  in  the  sputum  of  persons 
suffering  from  pulmonary  or  laryngeal  tuberculosis,  either  free  or  in 
the  interior  of  pus  cells  ;  in  miliary  tubercles  and  fresh  caseous 
masses,  in  the  lungs  or  elsewhere  ;  in  recent  tuberculous  cavities  in 
the  lungs ;  in  tuberculous  glands,  joints,  bones,  and  skin  affections 
(lupus) ;  in  the  lungs  of  cattle  suffering  from  pulmonary  tubercu- 
losis— Perlsucht ;  and  in  tubercular  nodules  generally  in  animals 
which  are  infected  naturally  or  by  experimental  inoculations. 

In  the  giant  cells  of  tubercular  growths  they  have  a  peculiar  and 
characteristic  position,  being  found,  as  a  rule,  upon  the  side  of  the 
cell  opposite  to  the  nuclei,  which  are  crowded  together  in  a  crescentic 
arrangement  at  the  opposite  pole  of  the  cell.  Sometimes  a  single 
bacillus  will  be  found  in  this  position,  or  there  may  be  several. 
Again,  numerous  bacilli  may  be  found  in  giant  cells  in  which  the 
nuclei  are  distributed  around  the  periphery.  They  are  more  numer- 
28 


394  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

ous  in  tuberculous  growths  of  recent  origin,  and  often  cannot  be 
demonstrated,  by  microscopical  examination,  in  caseous  material 
from  the  centre  of  older  nodules.  But  such  material,  when  inocu- 
lated into  susceptible  animals,  gives  rise  to  tuberculosis,  and  the 
usual  inference  is  that  it  contains  spores  of  the  tubercle  bacillus. 

Morphology. — The  tubercle  bacilli  are  rods  with  rounded  ends, 
of  from  1.5  to  3.5  //  in  length,  and  are  commonly  slightly  curved  or 

bent  at  an  angle ;  the  diameter  is 
about  0.2  p.  In  stained  preparations 
unstained  portions  are  frequently 
seen,  which  are  generally  believed  to 
be  spores,  but  this  is  by  no  means 
certain.  From  two  to  six  of  these 
unstained  spaces  may  often  be  seen 
in  a  single  rod,  and  owing  to  this  al- 
ternation of  stained  and  unstained 
portions  the  bacilli  may,  under  a  low 
power,  be  mistaken  for  chains  of  mi- 
crococci  The  rods  are  usually  soli- 

F,o.      US.  -  BaclHus      tuberculosis.       ^  but    "^  ^  United   in   PairS>  °r 

x  i ,000.  From  a  photomicrograph.  in  short  chains  containing  three  or  four 

elements.  In  old  cultures  irregular 

forms  may  be  observed,  the  rods  being  sometimes  swollen  at  one 
extremity,  or  presenting  the  appearance  of  having  a  lateral  bud-like 
projection — involution  forms. 

The  staining  characters  of  this  bacillus  are  extremely  important 
for  its  differentiation  and  recognition  in  preparations  of  sputum,  etc. 
Unlike  most  microorganisms  of  the  same  class,  it  does  not  readily 
take  up  the  aniline  colors,  and  when  stained  it  is  not  easily  decolorized, 
even  by  the  use  of  strong  acids.  The  failure  to  observe  it  in  tuber- 
culous material,  prior  to  Koch's  discovery,  was  no  doubt  due  to  the 
fact  that  it  does  not  stain  in  the  usual  aqueous  solutions  of  the  aniline 
dyes.  Koch  first  recognized  it  in  preparations  placed  in  a  staining 
fluid  to  which  an  alkali  had  been  added — solution  of  methylene  blue 
with  caustic  potash  ;  but  this  method  was  not  very  satisfactory,  and 
he  promptly  adopted  the  method  devised  by  Ehrlich,  which  consists 
essentially  in  the  use  of  a  solution  of  an  aniline  color — fuchsin  or 
methyl  violet — in  a  saturated  aqueous  solution  of  aniline  oil,  and  de- 
colorization  with  a  solution  of  a  mineral  acid — nitric  acid  one  part  to 
three  parts  of  water. 

The  original  method  of  Ehrlich  gives  very  satisfactory  results, 
but  various  modifications  have  since  been  proposed,  some  of  which 
are  advantageous.  The  carbol-fuchsin  solution  of  Ziehl  is  now 
largely  employed ;  it  has  the  advantage  of  prompt  action  and  of 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  395 

keeping  well.  The  staining  is  effected  more  quickly  if  heat  is  ap- 
plied. The  tubercle  bacilli  stain  by  Gram's  method,  but  this  is  not 
to  be  recommended  for  general  use,  owing  to  the  fact  that  the  pro- 
toplasm of  the  rods  is  frequently  contracted  into  a  series  of  spheri- 
cal, stained  bodies,  which  might  easily  be  mistaken  for  micrococci. 

The  examination  of  sputum  for  the  presence  of  the  tubercle  ba- 
cillus is  recognized  as  a  most  important  procedure  for  the  early  diag- 
nosis of  pulmonary  tuberculosis.     It  is  at- 
tended with  no  special  difficulties,  and  every 
physician  should  be  acquainted  with  the 
technique. 

The  patient  ehould  be  directed  to  expec- 
torate into  a  clean,  wide-mouthed  bottle  or 
glass-covered  jar  the  material  coughed  up 
from  the  lungs,  and  especially,  in  recent 
cases,  that  which  is  coughed  up  upon  first 
rising  in  the  morning.  This  should  be 
placed  in  the  physician's  hands  as  promptly  FIG.  117.— Bacillus  tubercuio- 
as  possible  ;  although  a  delay  of  some  days  f&^tum'  x  1'ooa>  (Baum' 
does  not  vitiate  the  result,  and  the  tubercle 

bacilli  may  still  be  demonstrated  after  the  sputum  has  undergone  pu- 
trefaction. It  is  well  to  pour  the  specimen  into  a  clean,  shallow  vessel 
having  a  blackened  bottom — a  Petri's  dish  placed  upon  a  piece  of  dead- 
black  paper  will  answer  very  well.  In  tuberculous  sputum  small,  len- 
ticular masses  of  a  yellowish  color  may  usually  be  observed,  and  one 
of  these  should  be  selected  for  microscopical  examination,  by  picking 
it  up  with  a  platinum  needle  and  freeing  it  as  far  as  possible  from 
the  tenacious  mucus  in  which  it  is  embedded.  If  such  masses  are 
not  recognized  take  any  purulent-looking  material  present  in  the 
specimen,  whether  it  be  in  small  specks  distributed  through  the  mu- 
cus, or  in  larger  masses.  A  little  of  the  selected  material  should  be 
placed  in  the  centre  of  a  clean  cover  glass  and  another  thin  glass 
cover  placed  over  it.  By  pressure  and  a  to-and-fro  motion  the  mate- 
rial is  crushed  and  distributed  as  evenly  as  possible  ;  the  glasses  are 
then  separated  by  a  sliding  motion.  The  film  is  permitted  to  dry  by 
exposure  in  the  air.  When  dry  the  cover  glass,  held  in  forceps,  is 
passed  three  times  through  the  flame  of  an  alcohol  lamp  or  Bunsen 
burner  to  fix  the  albuminous  coating.  Too  much  heat  causes  the  film 
to  turn  brown  and  ruins  the  preparation.  The  staining  fluid  (ZiehFs 
carbol-f  uchsin)  may  then  be  poured  upon  the  cover  glass,  or  this  may 
be  floated  upon  the  surface  of  the  fluid  contained  in  a  shallow  watch 
glass.  Heat  is  now  applied  by  bringing  the  cover  glass  over  a 
flame  and  holding  it  there  until  steam  begins  to  be  given  off  from 
the  surface  of  the  staining  fluid ;  it  is  then  withdrawn  and  again 


396  BACILLI   IN  CHRONIC  INFECTIOUS  DISEASES. 

gently  heated  at  intervals  for  a  minute  or  two.  The  cover  glass  is 
then  washed  in  water,  and  the  film  will  be  seen  to  have  a  uniform 
deep-red  color.  The  next  step  consists  in  decolorization  in  the  acid 
solution  (twenty-five-per-cent  solution  of  nitric  or  of  sulphuric  acid). 
The  cover  glass  is  gently  moved  about  in  this  solution  for  a  few 
seconds,  and  the  color  will  be  seen  to  quickly  fade  to  a  greenish 
tint.  The  object  is  to  remove  all  color  from  the  cells  and  the  al- 
buminous background,  so  that  the  bacilli,  which  retain  their  color  in 
presence  of  the  acid,  may  be  clearly  seen.  The  preparation  is  next 
washed  in  dilute  alcohol  (sixty  per  cent)  to  remove  the  fuchsin 
which  has  been  set  free  by  the  acid.  If  decolorization  was  not  car- 
ried far  enough  the  film  will  be  seen  to  still  have  a  red  color,  espe- 
cially in  places  where  it  is  thickest,  when  it  is  removed  from  the 
dilute  alcohol  and  washed  out  in  water.  In  this  case  it  will  be 
necessary  to  return  it  to  the  acid  solution  and  again  wash  it  in  the 
dilute  alcohol  and  in  water.  It  may  now  be  placed  in  a  solution 
of  methylene  blue  or  of  vesuvin  for  a  contrast  stain.  The  tubercle 
bacilli  are  distinguished  by  the  fact  that  they  retain  the  red  color 
imparted  to  them  in  the  fuchsin  solution,  while  other  bacteria  pre- 
sent, having  been  decolorized  in  the  acid  solution,  take  the  contrast 
stain  and  appear  blue  or  brown,  according  to  the  color  used.  The 
double-stained  preparation,  after  a  final  washing  in  water,  may  be 
examined  at  once,  or  dried  and  mounted  in  balsam  for  permanent 
preservation. 

Of  the  various  other  methods  which  have  been  proposed,  that  of 
Frankel,  as  modified  by  Gabbett,  appears  to  be  the  most  useful.  This 
consists  in  staining  as  above  directed  with  ZiehPs  carbol-f  uchsin  solu- 
tion, and  in  then  placing  the  cover  glass  directly  in  a  second  solution 
which  contains  both  the  acid  for  decolorizing  and  the  contrast  stain. 
This  second  solution  contains  twenty  parts  of  nitric  acid,  thirty  parts 
of  alcohol,  fifty  parts  of  water,  and  sufficient  methylene  blue  to  make 
a  saturated  solution  (one  to  two  parts  in  one  hundred).  After  re- 
maining in  this  solution  for  a  minute  or  two  the  cover  glass  is  washed 
in  water,  and  upon  microscopical  examination  the  tubercle  bacilli,  if 
present,  will  be  seen  as  red  rods  which  strongly  contrast  with  the 
blue  background. 

The  methods  recommended  for  cover-glass  preparations  may  also 
be  used  for  staining  the  tubercle  bacillus  in  thin  sections  of  tuber- 
culous tissues,  except  that  it  is  best  not  to  employ  heat.  The  sec- 
tions may  be  left  for  an  hour  in  the  carbol-f  uchsin  solution,  or  for 
twelve  hours  in  the  Ehrlich-Weigert  tubercle  stain— eleven  cubic 
centimetres  of  saturated  alcoholic  solution  of  methyl  violet,  ten  cubic 
(vntimi'tivsof  absolute  almlml,  <>n<>  hundred  cubictvntimctivs  of  ani- 
line water.  They  should  then  be  decolorized  by  placing  them  for 


BACILLI  IN   CHRONIC   INFECTIOUS   DISEASES. 


397 


about  half  a  minute  in  dilute  nitric  acid  (ten  per  cent) ;  then  wash 
out  color  in  sixty-per-cent  alcohol ;  counter-stain  for  two  or  three 
minutes  in  a  saturated  aqueous  solution  of  methylene  blue  ;  dehydrate 
with  absolute  alcohol  or  with  aniline  oil ;  clear  up  in  oil  of  cedar, 
and  mount  in  xylol  balsam.  If  the  aniline-water-methyl-violet  solu- 
tion has  been  used  for  staining  the  bacilli  a  saturated  solution  of 
vesuvin  may  be  used  as  a  contrast  stain. 

Biological  Characters. — A  parasitic,  aerobic,  non-motile  ba- 
cillus, which  grows  only  at  a  temperature  of  about  37°  C.  Is  also  a 
facultative  anaerobic  (Frankel). 

The  question  as  to  spore  formation  has  not  been  definitely  deter- 
mined. It  has  been  generally  assumed  that  the  unstained  spaces 
which  are  frequently  seen  in  the  bacilli  are  spores  ;  and  the  fact  that 


FIG.  118.— Section  through  a  tuberculous  nodule  in  the  lung  of  a  cow,  showing  two  giant  cells 
containing  tubercle  bacilli,  "x  950.    (Baftungarten.) 

caseous  material  in  which  a  microscopical  examination  has  failed  to 
demonstrate  the  presence  of  bacilli  may  produce  tuberculosis,  with 
bacilli,  when  inoculated  into  guinea-pigs,  has  been  explained  upon  the 
supposition  that  this  material  contained  spores.  But  a  few  bacilli 
present  in  such  caseous  material  might  easily  escape  detection.  As 
pointed  out  by  Frankel,  the  oval  spaces  in  stained  specimens  have 
not  the  sharply  denned  outlines  of  spores.  Moreover,  the  bacilli,  when 
examined  in  unstained  preparations,  do  not  contain  corresponding  re- 
fractive bodies,  recognizable  as  spores.  And  when  the  bacilli  are 
stained  by  Gram's  method  the  protoplasm  is  often  contracted  in  the 
form  of  little,  spherical  stained  masses,  while  the  unstained  spaces 
are  larger  and  no  longer  have  the  oval  form  presented  in  rods  stained 
by  Ehrlich's  method.  The  great  resisting  power  of  the  bacillus  to 
heat  and  to  desiccation  has  been  supposed  to  be  due  to  the  presence 


398  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

of  spores.  But,  so  far  as  resistance  to  heat  is  concerned,  this  is  not 
so  great  as  was  at  one  time  believed.  Schill  and  Fischer  (1884),  as- 
suming that  the  tubercle  bacillus  forms  spores,  made  quite  a  number 
of  experiments  to  determine  its  thermal  death-point.  They  sub- 
jected sputum  containing  the  bacillus  to  a  temperature  of  100°  C.,  and 
tested  the  destruction  of  vitality  by  inoculations  into  guinea-pigs. 
Exposure  to  steam  at  a  temperature  of  100°  C.  for  two  to  five  min- 
utes was  effective  in  every  experiment,  with  one  exception.  One 
guinea-pig  died  tuberculous  after  having  been  inoculated  with 
sputum  exposed  to  this  temperature  for  two  minutes.  This  result 
was  assumed  to  show  that  the  bacillus  would  survive  lower  tempera- 
tures, but  it  is  evident  that  additional  experiments  were  required  to 
establish  this  fact.  In  1887  the  writer  made  a  few  similar  experi- 
ments at  a  lower  temperature,  and  guinea-pigs  inoculated  with  tuber- 
culous sputum  exposed  for  ten  minutes  to  a  temperature  of  90°,  80°, 
and  60°  C.  failed  to  become  tuberculous,  while  another  guinea-pig, 
inoculated  with  the  same  material  after  exposure  to  a  temperature  of 
50°  C.  for  ten  minutes,  died  tuberculous.  These  results  correspond 
with  those  subsequently  (1888)  reported  by  Yersin,  who  tested  the 
thermal  death-point  of  this  bacillus  by  the  culture  method.  This 
author  assumes  that  the  bacilli  form  spores,  but  states  as  a  result  of 
his  experiments  that  "at  the  end  of  ten  days  bacilli  heated  for  ten 
minutes  at  55°  C.  gave  a  culture  in  glycerin-bouillon  ;  those  heated 
to  60°,  at  the  end  of  twenty-two  days;  while  those  heated  to  70°  and 
above  failed  to  grow  in  every  instance.  This  experiment,  repeated  a 
great  number  of  times,  always  gave  the  same  result.  The  tubercle 
bacilli  then  resist  a  temperature  of  60°  C.  for  ten  minutes,  and  it  is 
to  be  remarked  that  the  resistance  of  spores  to  heat  appears  to  be  no 
greater  than  that  of  the  bacilli  themselves/'  Yersin  remarks  in  a 
footnote  that  "the  spores  which  served  for  these  experiments  did 
not  appear  as  more  or  less  irregular  granules  taking  the  coloring 
matter  strongly,  but  as  veritable  spores  with  sharply  defined  outlines, 
to  the  number  of  one  or  two  in  a  bacillus,  or  three  at  the  outside. 
These  spores  are  particularly  clear  in  cultures  upon  glycerin-agar 
several  weeks  old." 

It  may  be  that  bacteriologists  have  been  mistaken  in  the  infer- 
ence that  all  spores  possess  a  greater  resisting  power  for  heat  than 
that  exhibited  by  bacilli  in  the  absence  of  spores.  That  this  is  true 
as  iv^anls  anthrax  spores  ami  many  others,  tlio  thermal  death-point 
of  which  has  been  determined  by  exact  experiments,  does  not  prove 
that  it  is  true  for  all.  And  it  is  known  that  there  are  wide  differ- 
ences in  the  resisting  power  both  of  the  spores  of  different  species 
and  in  the  vegetating  cells.  To  admit  that  the  tubercle  bacillus  or 
the  typhoid  bacillus,  etc.,  may  form  spores  which  have  no  greater 


BACILLI   IN   CHRONIC   INFECTIOUS  DISEASES.  399 

resisting  power  against  heat  than  the  bacilli  themselves,  would  there- 
fore simply  be  an  admission  that  some  bacteriologists  had  made  a 
mistaken  inference  based  upon  incomplete  data.  In  view  of  the 
facts  stated  we  can  simply  repeat  what  was  said  at  the  outset,  viz. , 
the  question  as  to  spore  formation  has  not  been  definitely  deter- 
mined. 

The  tubercle  bacillus  is  a  strict  parasite,  and  its  biological  char- 
acters are  such  that  it  could  scarcely  find  natural  conditions,  outside 
of  the  bodies  of  living  animals,  favorable  for  its  multiplication.  It 
therefore  does  not  grow  as  a  saprophyte  under  ordinary  circum- 
stances. But  it  has  been  noted  by  Roux  and  Nocard  that  when  it 
has  been  cultivated  for  a  time  in  artificial  media  containing  glycerin 
it  may  grow  in  a  plain  bouillon  of  veal  or  chicken,  in  which  media  it 
fails  to  develop  when  introduced  directly  from  a  culture  originating 
from  the  body  of  an  infected  animal.  This  would  indicate  the  pos- 
sibility of  its  acquiring  the  ability  to  grow  as  a  saprophyte  ;  and  we 
can  scarcely  doubt  that  at  some  time  in  the  past  it  was  a  true  sapro- 
phyte. The  experiments  of  Nuttall  indicate  that  the  bacillus  may 
multiply,  under  favorable  temperature  conditions,  in  tuberculous 
sputum  outside  of  the  body.  And  it  is  extremely  probable  that  mul- 
tiplication occurs  in  the  muco-purulent  secretion  which  accumulates 
in  pulmonary  cavities  in  phthisical  patients.  In  these  cavities  its  de- 
velopment may,  in  a  certain  sense,  be  regarded  as  saprophytic,  as  it 
feeds  upon  non-living  organic  material. 

Koch  first  succeeded  in  cultivating  this  bacillus  upon  coagulated 
blood  serum,  prepared  as  directed  in  Section  VIII.,  Part  First,  of  the 
present  volume.  Roux  and  Nocard  have  since  shown  (1888)  that  it 
grows  very  well  on  nutrient  agar  to  which  glycerin  has  been  added 
(six  to  eight  per  cent),  and  also  in  veal  broth  containing  five  per  cent 
of  glycerin.  It  is  difficult  to  obtain  pure  cultures  from  tuberculous 
sputum,  on  account  of  the  presence  of  other  bacteria  which  grow 
much  more  rapidly  and  take  full  possession  of  the  medium  before  the 
tubercle  bacillus  has  had  time  to  form  visible  colonies,  l^or  this  rea- 
son it  is  best  to  first  inoculate  a  guinea-pig  with  the  tuberculous  spu- 
tum and  to  obtain  cultures  from  it  after  tuberculous  infection  has 
fully  developed.  The  inoculated  animals  usually  die  at  the  end  of 
three  or  four  weeks.  It  is  best  to  kill  one  which  gives  evidence  of 
being  tuberculous,  and  to  remove  one  or  more  nodules  from  the 
lungs  through  an  opening  made  in  the  chest  walls.  The  greatest 
care  will  be  required  to  prevent  contamination  by  other  common 
microorganisms.  The  instruments  used  must  be  sterilized  by  heat, 
and  the  skin  over  the  anterior  thoracic  wall  carefully  turned  back  ; 
then,  after  again  sterilizing  knives  and  scissors,  cut  an  opening  into 
the  chest  cavity,  draw  out  the  root  of  the  lung,  and  take  up  with 


400  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

slender  sterilized  forceps,  or  with  a  strong  platinum  loop,  one  or 
more  well-defined  tubercular  nodules.  These  may  be  conveyed  di- 
rectly to  the  surface  of  the  solid  culture  medium  and  then  broken 
up  and  rubbed  over  the  surface  as  thoroughly  as  possible  ;  or  they 
may  first  be  crushed  between  two  sterilized  glass  slides,  and  then 
transferred  with  the  platinum  loop  and  thoroughly  rubbed  into  the 
surface  of  the  culture  medium. 

This  breaking-up  of  the  tuberculous  nodules  and  distribution  of 
the  bacilli  upon  the  surface  of  the  culture  medium  is  essential  for 
the  success  of  the  experiment.  Instead  of  using  the  tubercular 
nodules  in  the  lungs,  an  enlarged  lymphatic  gland  from  the  axilla  or 
elsewhere  may  be  used,  as  first  recommended  by  Koch.  This  is  to 
be  crushed  in  the  same  way ;  and  it  will  be  best  to  inoculate  a  num~ 
her  of  tubes  at  the  same  time,  as  accidental  contamination  or  failure 
to  develop  is  very  liable  to  occur  in  a  certain  number.  Owing  to  the 
liability  of  the  blood  serum  to  become  too  dry  for  the  development  of 
the  bacillus,  it  is  best  to  keep  the  cultures  in  a  moist  atmosphere,  or 
to  prevent  evaporation  by  applying  a  rubber  cap  over  the  open  end 
of  the  test  tube.  This  should  be  sterilized  in  a  solution  of  mercuric 
chloride  (1  : 1,000) ;  and  the  end  of  the  cotton  plug  should  be  burned 
off  just  before  applying  it,  for  the  purpose  of  destroying  the  spores 
of  mould  fungi,  which  in  a  dry  atmosphere  would  be  harmless,  but 
under  the  rubber  cap  are  likely  to  sprout  and  to  send  their  mycelium 
through  the  cotton  plug  to  the  interior  of  the  tube,  thus  destroying 
the  culture. 

Upon  coagulated  blood  serum  the  growth  first  becomes  visible  at 
the  end  of  ten  to  fourteen  days  (at  37°  0.),  and  at  the  end  of  three 
weeks  a  very  distinct  and  characteristic  develop- 
ment has  occurred.    The  first  appearance  is  that  of 
dry-looking,  grayish-white  points  and  scales,  which 
are  without  lustre,  and  are  sometimes  united  to 
form  a  thin,  irregular,  membranous-looking  layer. 
Under  the  microscope,  with  an  amplification  of 
eighty  diameters,  the  early,  thin  surface  growth 
upon  blood  serum  presents  a  characteristic  appear- 
ance.   The  bacilli,  arranged  in  parallel  rows,  form 
variously  curved  figures,  of  which  we  may  obtain 
impressions  by  carefully  applying  a  dry  cover  glass 
< mi ureupon blood  se-       to  the  surface.     Upon  staining  the  preparation  in 
the  usual  way  the  same  arrangement  of  the  bacilli 
which  adhered  to  the  thin  glass  cover  will  be  pre- 
served.    The    growth  is  more   abundant    in  subsequent  cultures, 
which  have  Ix-m    kept  up  in  Koch's  laboratory  from  his  original 
pure  cultures  up  to  the  present  time  ;  in  these  the  bacillus  still   pre- 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  401 

serves  its  characters  of  form  and  growth,  and  its  specific  pathogenic 
power. 

Pastor  (1892)  has  succeeded  in  obtaining  pure  cultures  of  the 
tubercle  bacillus  from  sputum  by  the  following  ingenious  method  : 
After  proving  by  microscopic  examination  that  the  sputum  of  a 
tuberculous  individual  contains  numerous  bacilli,  he  has  the  patient 
cleanse  his  mouth  as  thoroughly  as  possible  with  sterilized  water, 
and  then  expectorate  some  material,  coughed  up  from  the  lungs,  into 
a  sterilized  test  tube.  By  shaking  with  sterilized  water  a  fine  emul- 
sion is  made,  and  this  is  filtered  through  fine  gauze.  The  filtrate, 
which  is  nearly  transparent,  contains  numerous  tubercle  bacilli.  A 
few  drops  of  the  emulsion  are  now  added  to  liquefied  gelatin  in  a  test 
tube,  and  a  plate  is  made  in  the  usual  way.  This  is  kept  for  three 
or  four  days  at  the  room  temperature,  during  which  time  the  com- 
mon mouth  bacteria  capable  of  growth  form  visible  colonies.  By 
means  of  a  hand  lens  a  place  is  now  selected  in  which  no  colonies  are 
seen,  and  a  bit  of  gelatin  is  excised  with  a  sterilized  knife.  This 
piece  is  transferred  to  the  surface  of  blood  serum  or  glycerin-agar, 
and  placed  in  the  incubating  oven,  where  in  due  time  colonies  of 
the  tubercle  bacillus  will  usually  be  found  to  develop. 

Another  method  of  accomplishing  the  same  result  has  recently 
been  described  by  Kitasato.  This  is  a  method  devised  by  Koch  some 
time  since  and  successfully  employed  in  his  laboratory.  The  morn- 
ing expectoration  of  a  tuberculous  patient,  raised  from  the  lungs  by 
coughing,  is  received  in  a  Petri/s  dish.  A  bit  of  sputum,  such  as 
comes  from  the  tuberculous  cavity  in  the  lungs  of  such  a  patient,  is 
now  isolated  with  sterilized  instruments  and  carefully  washed  in  at 
least  ten  successive  portions  of  sterilized  water.  By  this  procedure 
the  bacteria  accidentally  attached  to  the  viscid  mass  of  sputum  dur- 
ing its  passage  through  the  mouth  are  washed  away.  In  the  last 
bath  the  mass  is  torn  apart  and  a  small  portion  from  the  interior  is 
used  to  make  a  microscopic  preparation,  the  examination  of  which 
shows  whether  only  tubercle  bacilli  are  present.  If  this  be  the  case 
cultures  upon  glycerin-agar  are  started  from  material  obtained  from 
the  interior  of  the  same  mass.  The  colonies  obtained  in  this  way 
appear  in  about  two  weeks  as  round,  white,  opaque,  moist,  and  shin- 
ing masses.  Kitasato's  researches  show  that  the  greater  portion  of 
the  tubercle  bacilli  in  sputum  obtained  in  this  way,  and  in  the  con- 
tents of  lung  cavities,  are  incapable  of  development,  although  this 
fact  cannot  be  recognized  by  a  microscopic  examination  of  stained 
specimens. 

On  account  of  the  greater  facility  of  preparing  and  sterilizing 
glycerin-agar,  and  the  more  rapid  and  abundant  development  upon 
this  medium,  it  is  now  usually  employed  in  preference  to  blood 


402 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


Serum.  The  growth  at  the  end  of  fourteen  days  is  more  abundant  than 
upon  blood  serum  at  the  end  of  several  weeks.  When  numerous 
bacilli  have  been  distributed  over  the  surface  of  the  culture  medium 
a  rather  uniform,  thick,  white  layer,  which  subsequently  acquires  a 
yellowish  tint,  is  developed  ;  when  the  bacilli 
are  few  in  number  or  are  associated  in  scattered 
groups  separate  colonies  are  developed,  which 
acquire  considerable  thickness  and  have  more 
or  less  irregular  outlines  ;  they  are  white  at 
first,  then  yellowish-white.  Frankel  describes 
the  tubercle  bacillus  as  a  facultative  anaerobic, 
and  it  would  appear  that  it  must  be  able  to  grow 
in  situations  where  it  can  obtain  very  little  oxy- 
gen from  its  development  in  the  interior  of  tu- 
berculous nodules,  lymphatic  glands,  etc.  But 
in  stick  cultures  in  glycerin-agar  development 
only  occurs  near  the  surface,  and  not  at  all  in 
the  deeper  portion  of  the  medium.  In  view  of 
its  abundant  growth  on  the  surface  it  is  diffi- 
cult to  understand  this  failure  to  grow  along 
the  line  of  puncture,  if  it  is  in  truth  a  faculta- 
tive anaerobic. 

In  peptonized  veal  broth  containing  five  per 
cent  of  glycerin  the  bacillus  develops  at  first  in 
the  form  of  little  flocculi,  which  accumulate  at 
the  bottom  of  the  flask  and  which  by  agitation 
are  easily  broken  up.  At  the  end  of  two  or 
three  weeks  the  bottom  of  the  flask  is  covered 
with  similar  flocculi,  which  form  an  abundant 
deposit. 

Pawlowski  and  others  report  success  in  cul- 
tivating the  tubercle  bacillus  upon  the  surface 
of  cooked  potato  enclosed  in  a  test  tube  after 
the  method  of  Bolton  and  Koux.  The  open  end 
of  the  tube  is  hermetically  sealed  in  a  flame 
after  the  bacilli  have  been  planted  upon  the 
obliquely-cut  surface  of  the  potato;  this  prevents  drying.  Ac- 
cording to  Pawlowski,  better  results  are  obtained  if  the  surface  of 
the  potato  is  moistened  with  a  five-per-cent  solution  of  glycerin.  The 
growth  is  said  to  be  seen  at  the  end  of  about  twelve  days  as  grayish, 
diy-looking  flakes  ;  at  the  end  of  three  or  four  weeks  it  forms  a  dry, 
smooth,  whitish  layer,  and  no  further  development  occurs. 

The  range  of   temperature  at  which  this  bacillus  will  grow  is 
very  restricted  ;  37°  C.  is  usually  given  as  the  most  favorable  point, 


Fio.  120.— Culture  of  tu- 
bercle bacillus  upon  glyce- 
rin-agon Photograph  by 
Roux. 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  403 

but  Roux  and  Nocard  say  that  the  most  favorable  temperature  ap- 
pears to  be  39°,  and  that  development  is  slower  at  37°. 

The  experiments  of  Koch,  Schill  and  Fischer,  and  others  show 
that  the  bacilli  retain  their  vitality  in  desiccated  sputum  for  several 
months  (nine  to  ten  months — De  Toma) ;  but  they  are  said  to  undergo 
a  gradual  diminution  in  pathogenic  virulence,  which  is  more  rapid 
when  the  desiccated  material  is  kept  at  a  temperature  of  30°  to  40°  C. 
In  the  experiments  of  Cadeac  and  Malet  portions  of  the  lung  from 
a  tuberculous  cow,  dried  and  pulverized,  produced  tuberculosis  in 
guinea-pigs  at  the  end  of  one  hundred  and  two  days.  They  retain 
their  vitality  for  a  considerable  time  in  putrefying  material  (forty- 
three  days — Schill  and  Fischer  ;  one  hundred  and  twenty  days — Ca- 
deac and  Malet).  The  resisting  power  of  this  bacillus  against  ger- 
micidal  agents  is  also  greater  than  that  of  certain  other  pathogenic 
microorganisms,  but  not  so  great  as  to  justify  the  inference  that  it 
forms  spores.  It  is  not  destroyed  by  the  gastric  juice  in  the  sto- 
mach, as  is  shown  by  successful  infection  experiments  in  suscep- 
tible animals,  by  mixing  cultures  of  the  bacillus  with  their  food 
(Baumgarten,  Fischer),  and  also  by  experiments  with  an  artificially 
prepared  gastric  juice  (Falk).  They  are  destroyed,  in  sputum,  in 
twenty  hours  by  a  three-per-cent  solution  of  carbolic  acid,  even 
when  they  present  the  appearance  usually  ascribed  to  the  presence 
of  spores  (Cavagnis)  ;  also  by  absolute  alcohol,  a  saturated  aqueous 
solution  of  salicylic  acid,  saturated  aniline  water,  etc.  (Schill  and 
Fischer).  The  more  recent  experiments  of  Yersin  upon  pure  cul- 
tures of  the  bacillus  gave  the  following  results  :  "  Tubercle  bacilli, 
containing  spores,  were  killed  by  a  five-per-cent  solution  of  carbolic 
acid  in  thirty  seconds,  by  one-per-cent  in  one  minute  ;  absolute  alco- 
hol, five  minutes  ;  iodof orm-ether,  one  per  cent,  five  minutes  ;  ether, 
ten  minutes ;  mercuric  chloride,  1  : 1,000  solution,  ten  minutes ; 
thymol,  three  hours  ;  salicylic  acid,  2. 5  per  cent,  six  hours. 

The  tubercle  bacillus  appears  to  be  especially  susceptible  to  the 
action  of  light.  In  his  address  before  the  Tenth  International  Medi- 
cal Congress  (Berlin,  1890)  Koch  says  that  when  exposed  to  direct 
sunlight  the  tubercle  bacillus  is  killed  in  from  a  few  minutes  to  sev- 
eral hours,  according  to  the  thickness  of  the  layer ;  it  is  also  de- 
stroyed by  diffuse  daylight  in  from  five  to  seven  days  when  placed 
near  a  window.  This  fact  has  an  important  hygienic  bearing,  espe- 
cially in  view  of  the  fact  that  the  tubercle  bacillus  is  not  readily 
killed  by  desiccation,  putrefaction  of  the  material  containing  it,  etc. 
Tuberculous  sputum  expectorated  upon  sidewalks,  etc.,  being  ex- 
posed to  the  action  of  direct  sunlight,  will  in  many  cases  be  disin- 
fected by  this  agent  by  the  time  complete  desiccation  has  occurred — 
i.  e. ,  before  it  is  in  a  condition  to  be  carried  into  the  air  as  dust. 


404  BACILLI  IN  CHRONIC   INFECTIOUS  DISEASES. 

Sawizky  in  1891  made  a  series  of  experiments  to  determine 
the  length  of  time  during  which  d  ried  tuberculous  sputum  retains 
its  virulence.  He  arrived  at  the  conclusion  that  virulence  is  not  sud- 
denly but  gradually  lost,  and  that  in  an  ordinary  dwelling  room 
dried  sputum  retains  its  specific  infectious  power  for  two  and  one- 
half  months. 

Metschnikoff  states  that  when  kept  at  a  temperature  of  42°  C.  for 
some  time  the  tubercle  bacillus  undergoes  a  notable  diminution  in 
its  pathogenic  power,  and  that  when  kept  at  a  temperature  of  43°  to 
44°  it  after  a  time  only  induces  a  local  abscess  when  injected  subcu- 
taneously  into  guinea-pigs.  The  experiments  of  Lote  also  indicate 
that  an  "  attenuation  of  virulence  "  has  occurred  in  the  cultures  pre- 
served in  Koch's  laboratory,  originating  in  1882  from  the  lungs  of  a 
tuberculous  ape.  The  author  named  made  experiments  with  cul- 
tures from  this  source  (ninetieth  to  ninety-fifth  successive  culture), 
and  at  the  same  time  with  a  culture  obtained  from  Roux,  of 
Pasteur's  laboratory.  Rabbits  inoculated  with  cultures  from  the 
last-mentioned  source  developed  a  hectic  fever  at  the  end  of  two 
weeks,  and  died  tuberculous  at  the  end  of  twenty-one  to  thirty-nine 
days.  Twelve  rabbits  were  inoculated  with  the  cultures  from 
Koch's  laboratory  ;  the  injections  were  made  either  subcutaneously, 
into  a  vein,  into  the  pleural  cavity,  or  into  the  cavity  of  the  abdo- 
men. No  elevation  of  temperature  occurred  in  any  of  the  animals, 
and  they  were  found  at  the  end  of  a  month  to  have  increased  in 
weight.  At  the  end  of  six  weeks  one  of  them  was  killed  and  tuber- 
cular nodules  were  found  in  various  organs.  The  remaining  animals 
were  killed  at  the  end  of  one  hundred  and  forty-four  to  one  hundred 
and  forty-eight  days.  The  two  inoculated  subcutaneously  presented 
no  sign  of  general  tuberculosis,  but  a  small  yellow  nodule  contain- 
ing bacilli  was  found  at  the  point  of  inoculation.  Those  inoculated 
by  injection  into  a  vein  showed  one  or  two  nodules  in  the  lungs  con- 
taining a  few  bacilli.  In  Koch's  original  experiments  rabbits  were 
killed  by  intravenous  inoculation  of  his  cultures  in  from  thirteen  to 
thirty-one  days.  That  this  attenuation  of  virulence  depends  upon  a 
<liminished  production  of  a  toxic  product  to  which  the  bacillus  owes 
its  pathogenic  power  appears  to  be  very  certain,  in  view  of  the  fact 
that  the  late  cultures  in  a  series  have  a  more  vigorous  and  abundant 
development  than  the  more  pathogenic  cultures  obtained  directly 
f  r<  »in  the  animal  body. 

The  discovery  by  Koch  of  a  toxine  in  cultures  of  this  bacillus, 
which  is  soluble  in  glycerin,  and  which  in  very  minute  doses  pro- 
duces febrile  reaction  and  other  decided  symptoms  when  injected  sub- 
cutaneously into  tuberculous  animals,  must  rank  as  one  of  the  first 


BACILLI   IN"   CHRONIC   INFECTIOUS   DISEASES.  405 

importance  in  scientific  medicine,  whatever  the  final  verdict  may  be 
as  to  its  therapeutic  value  in  tubercular  diseases  in  man. 

The  toxic  substance  contained  in  Koch's  glycerin  extract  from 
cultures  of  the  tubercle  bacillus,  now  generally  known  under  the 
name  of  tuberculin,  is  soluble  in  water,  insoluble  in  alcohol,  and 
passes  readily  through  dialyzing  membranes.  It  is  not  destroyed  by 
the  boiling  temperature.  According  to  the  chemical  examination  of 
Jolles,  the  "  lymph  "  contains  fifty  per  cent  of  water  and  does  not 
contain  alkaloids  or  cyanogen  compounds.  It  contains  albuminates, 
which  are  thrown  down  as  a  voluminous  white  precipitate  by  tannic 
acid,  and  are  redissolved  by  hot  water  containing  sodium  chloride 
and  very  diluted  potash  solution.  The  elementary  analysis  gave 
1ST  5.90  per  cent,  C  35.19  per  cent,  and  H  7.02  per  cent.  The  re- 
sults obtained  are  believed  to  show  that  the  active  substance  present 
in  the  lymph  is  a  toxalbumin.  In  experiments  made  with  Koch's 
lymph  in  Pasteur's  laboratory  by  Bardach,  a  very  decided  elevation 
of  temperature  was  produced  in  tuberculous  guinea-pigs  by  the  sub- 
cutaneous injection  of  0.1  gramme,  and  a  fatal  result  by  the  injec- 
tion of  0. 2  to  0. 5  gramme.  In  man  a  decided  febrile  reaction  is  pro- 
duced in  tuberculous  patients  by  very  much  smaller  doses — 0.001 
cubic  centimetre. 

Hammerschlag,  in  his  chemical  researches,  found  that  the  tubercle 
bacillus  yields  a  larger  proportion  of  substances  soluble  in  alcohol 
and  ether  than  any  other  bacilli  tested  (twenty-seven  per  cent).  The 
alcoholic  extract  contains  fat,  lecithin,  and  a  toxic  substance  which 
produces  convulsions  in  rabbits  and  guinea-pigs.  The  portion  in- 
soluble in  alcohol  and  ether  contains  cellulose  and  an  albuminoid 
substance.  No  ptomaines  were  found,  but  a  toxalbumin  was  isolated, 
which  caused  an  elevation  of  temperature  in  rabbits  of  1°  to  2°  C., 
lasting  for  a  day  or  two. 

Hunter  reports  the  following  results  of  his  chemical  examination 
of  tuberculin.  It  contains — 

1.  Albumoses,  chiefly  protoalbumose  and  deuteroalbumose,  along  with 
heteroalbumose,  and  occasionally  a  trace  of  dysalbumose. 

2.  Alkaloidal  substances,  two  of  which  can  be  obtained  in  the  form  of 
the  platinum  compounds  of  their  hydrochlorate  salts. 

3.  Extractives,  small  in  quantity  and  of  unrecognized  nature. 

4.  Mucin. 

5.  Inorganic  salts. 

6.  Glycerin  and  coloring  matter. 

The  following  conclusions  are  reached  with  reference  to  its  toxic 
properties  : 

1.  Tuberculin  owes  its  activity,  not  to  one  principle,  but  to  at  least  three, 
and  probably  more,  different  substances. 

2.  Its  action  in  producing  local  inflammation,  fever,  and  general  consti- 
tutional disturbance  is  not  a  simple  but  an  extremely  complex  one. 


406  BACILLI  IN  CHRONIC   INFECTIOUS  DISEASES. 

3.  Its  active  ingredients  are  of  the  nature  of  albumoses,  alkaloidal  sub- 
stances, and  extractives.    The  action  of  these  is  in  certain  instances  antag- 
onistic. 

4.  Its  remedial  and  inflammatory  actions  are  connected  with  the  presence 
of  certain  of  its  albumoses,  while  its  fever  producing  properties  are  chiefly 
associated  with  substances  of  non-albuminous  nature. 

5.  The  albumoses  are  not  lost  by  dialysis ;  the  latter  are.    By  the  adoption 
of  suitable  methods  it  is  thus  possible  to  remove  the  substances  which  cause 
the  fever,  while  retaining  those  which  are  beneficial  in  their  action. 

6.  The  fever  produced  by  tuberculin  is  thus  absolutely  unessential  to  its 
remedial  action. 

In  a  communication  dated  October,  1891,  Koch  has  given  a  full 
account  of  his  method  of  preparing  crude  tuberculin,  and  also  the 
process  by  which  he  obtains  from  this  a  tuberculin  which  appears  to 
be  pure,  or  nearly  so.  To  obtain  considerable  quantities  of  the  crude 
product  the  tubercle  bacillus  is  cultivated  in  an  infusion  of  calves' 
flesh  or  of  beef  extract  to  which  one  per  cent  of  peptone  and  four  to 
five  per  cent  of  glycerin  have  been  added.  This  culture  liquid  must 
be  made  slightly  alkaline,  and  it  is  placed  in  flasks  with  a  flat  bottom, 
which  should  not  be  more  than  half -filled — thirty  to  fifty  cubic  centi- 
metres. The  inoculation  is  made  upon  the  surface  with  small  masses 
from  a  culture  upon  blood  serum  or  glycerin-agar.  By  accident 
Koch  discovered  that  these  masses  floating  upon  the  surface  give  rise 
to  an  abundant  development  and  to  the  formation  of  a  tolerably  thick 
and  dry  white  layer,  which  finally  covers  the  entire  surface.  At  the 
end  of  six  to  eight  weeks  development  ceases  and  the  layer  after  a 
time  sinks  to  the  bottom,  breaking  up  meanwhile  into  fragments. 
These  cultures,  after  their  purity  has  been  tested  by  a  microscopical 
examination,  are  poured  into  a  suitable  vessel  and  evaporated  to  one- 
tenth  the  original  volume  over  a  water  bath.  The  liquid  is  then  fil- 
tered through  porcelain.  The  crude  tuberculin  obtained  by  this  pro- 
cess contains  from  forty  to  fifty  per  cent  of  glycerin,  and  consequently 
is  not  a  suitable  medium  for  the  development  of  saprophytic  bacteria, 
if  they  should  by  accident  be  introduced  into  it.  It  keeps  well  and 
preserves  its  activity  indefinitely. 

From  this  crude  tuberculin  Koch  has  obtained  a  white  precipitate, 
with  sixty-per-cent  alcohol,  which  has  the  active  properties  of  the 
crude  tuberculin  as  originally  prepared.  This  is  fatal  to  tuberculous 
guinea-pigs  in  doses  of  two  to  ten  milligrammes.  It  is  soluble  in 
water  and  in  glycerin,  and  has  the  chemical  reactions  of  an  albu- 
minous  body.  In  preparing  it  one  and  a  half  volumes  of  absolute 
alcohol  are  added  to  one  volume  of  the  crude  tuberculin,  and,  after 
stirring  it  to  secure  uniform  admixture,  this  is  put  aside  for  twenty-four 
hours.  At  the  end  of  this  time  a  flocculent  deposit  will  be  seen  at  the 
bottom  of  the  vessel.  The  fluid  above  this  is  carefully  poured  off, 
and  an  equal  quantity  of  sixty-per-cent  alcohol  poured  into  the  vessel 


BACILLI    IN   CHRONIC   INFECTIOUS   DISEASES.  407 

for  the  purpose  of  washing  the  precipitate.  This  is  again  allowed  to 
settle  and  the  procedure  is  repeated  three  or  four  times,  after  which 
the  precipitate  is  washed  with  absolute  alcohol.  It  is  then  placed 
upon  a  filter  and  dried  in  a  vacuum  exsiccator. 

An  analysis  of  this  purified  tuberculin  by  Proskauer  gave  18.46 
per  cent  of  ash,  consisting  almost  entirely  of  potassium  and  magne- 
sium phosphate.  The  elementary  analysis  gave  48.13  per  cent  of 
carbon,  7.06  per  cent  of  hydrogen,  14.46  per  cent  of  nitrogen,  and 
1.17  per  cent  of  sulphur. 

Tizzoni  and  Cattani  (1892)  have  presented  some  experimental  evi- 
dence which  indicates  that  injections  of  Koch's  tuberculin  into 
guinea-pigs  may  produce  in  these  animals  a  certain  degree  of  im- 
munity against  tuberculosis ;  and  that  this  immunity  depends  upon 
the  presence  of  an  anti-tuberculin  formed  in  the  body  of  the  partially 
immune  animal. 

Numerous  experiments  made  by  veterinary  surgeons  upon  tuber- 
culous cows  show  that  the  injection  of  Koch/s  tuberculin  in  these 
animals,  in  doses  of  thirty  to  forty  centigrammes,  produces  a  rise  of 
temperature  of  from  1°  to  3°  C.  The  febrile  reaction  usually  occurs 
in  from  twelve  to  fifteen  hours  after  the  injection.  Its  duration  and 
intensity  do  not  depend  upon  the  extent  of  the  tuberculous  lesions, 
but  is  even  more  marked  when  these  are  slight  than  in  advanced 
cases.  In  non-tuberculous  animals  no  reaction  occurs,  and  the  ex- 
periments made  justify  the  suspicion  that  tuberculosis  exists  if  an. 
elevation  in  temperature  of  a  degree  or  more  occurs  as  a  result  of 
the  subcutaneous  injection  of  the  dose  mentioned. 

When  the  number  of  tubercle  bacilli  in  sputum  is  comparatively 
small  they  may  easily  escape  observation.  Methods  have  therefore 
been  suggested  for  finding  them  under  these  circumstances.  Ribbert 
(1886)  proposed  the  addition  to  the  sputum  of  a  two-per-cent  solution 
of  caustic  potash,  and  boiling  the  mixture.  The  tenacious  mucus  is 
dissolved,  and  when  the  mixture  is  placed  in  a  conical  glass  vessel 
the  bacilli  are  deposited  at  the  bottom  and  may  easily  be  found  in 
the  sediment  after  removing  the  supernatant  fluid.  The  same  object 
is  accomplished  by  Stroschein  (1889)  by  the  addition  to  sputum  of 
three  times  its  volume  of  a  saturated  solution  of  borax  and  boracic 
acid  in  water. 

A  method  of  estimating  the  number  of  bacilli  in  sputum  has  re- 
cently been  proposed  by  Nuttall,  which  appears  to  give  sufficiently 
accurate  results  and  to  be  useful  in  judging  of  the  progress  of  a 
case  or  of  the  results  of  treatment.  For  the  details  of  this  method 
we  must  refer  to  the  author's  paper  (Johns  Hopkins  Hospital  Bulle- 
tin, vol.  xi.?  No.  13,  1891).  It  consists  essentially  in  first  making 
the  sputum  fluid  by  the  addition  of  a  solution  of  caustic  potash  ;  in 


408 


BACILLI  IN   CHRONIC   INFECTIOUS   DISEASES. 


then  shaking  it  thoroughly  in  a  bottle  containing  sterilized  gravel 
or  pounded  glass  ;  in  carefully  measuring  the  total  quantity  of  fluid, 
and  in  dropping  upon  glass  slides  uniform  drops  by  means  of  a  grad- 
uated pipette  ;  in  spreading  these  uniformly  by  means  of  a  platinum 
needle  and  a  turn  table ;  in  covering  the  dried  film  with  a  film  of 
blood  serum,  and  coagulating  this  by  heat ;  and,  finally,  in  staining 
and  counting  the  bacilli  in  a  series  of  slides  from  the  same  specimen, 
and  from  the  average  number  found  in  a  single  drop  estimating  the 
total  number  in  the  sputum  for  twenty-four  hours. 

Pathogenesis. — Man,  cattle,  and  monkeys  are  most  subject  to 
contract  the  disease  naturally,  and  it  may  be  communicated  by  in- 
oculation to  many  of  the  lower  animals — guinea-pigs,  field  mice,  rab- 


Fio.  121,-Llmited  epithelioid  celled  tubercle  of  the  iris,    x  950.    (Baumgarten.) 

bits,  and  cats  are  among  the  most  susceptible  animals  ;  and  in  larger 
doses  dogs,  rats,  white  mice,  and  fowls  may  also  be  infected. 

When  tuberculous  sputum  is  introduced  beneath  the  skin  of  a 
K'liiiea-pig  the  nearest  lymphatic  glands  are  found  to  be  swollen  at 
the  end  of  two  or  three  weeks,  at  the  same  time  there  is  a  thickening 
of  the  tissues  about  the  point  of  inoculation  ;  later  a  dry  crust  forms 
over  the  local  tuberculous  tumefaction,  and  beneath  this  is  a  flattened 
ulcer  covered  with  cheesy  material.  The  animals  become  emaciated 
and  sh«>\v  ditliculty  in  breathing  jind  usually  succumb  to  general 
tuberculosis,  especially  involving  the  lungs,  within  four  to  eight 
weeks,  Injections  of  tuberculous  sputum,  or  of  pure  cultures  of  the 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  409 

bacillus,  into  the  peritoneal  cavity  give  rise  to  extensive  tuberculo- 
sis of  the  liver,  spleen,  and  lungs,  and  to  death,  as  a  rule,  within 
three  or  four  weeks.  Rabbits  are  less  susceptible  to  subcutaneous 
injections,  but  die  within  seventeen  to  twenty  days  when  virulent — 
recent — cultures  are  injected  into  the  circulation.  As  a  result  of 
such  an  inoculation  the  animal  rapidly  loses  flesh  and  has  a  decided 
elevation  of  temperature,  commencing  at  the  end  of  the  first  week 
and  increasing  considerably  during  the  last  days  of  life.  At  the 
autopsy  the  spleen  and  liver  are  found  to  be  greatly  enlarged,  but 
they  do  not  contain  any  tubercles  that  can  be  recognized  by  the  naked 
eye  (Yersin).  They  contain,  however,  great  numbers  of  tubercle 
bacilli,  both  free  and  in  the  cells.  Injections  of  a  small  quantity  of 
a  pure  culture  into  the  anterior  chamber  of  the  rabbit's  eye  cause 
first  iris-tuberculosis,  followed  by  swelling  and  caseation  of  the  near- 
est lymph  glands,  and  finally  general  infection  and  death ;  when 
larger  quantities  are  injected  general  tuberculosis  is  quickly  devel- 
oped. The  influence  of  quantity — number  of  bacilli — is  also  shown 
in  subcutaneous,  intravenous,  or  intraperitoneal  injections  into  guinea- 
pigs  and  rabbits  (Hirschberger,  Gebhardt,  Wyssokowitsch).  Thus 
rabbits  which  received  less  than  one  hundred  and  fifty  bacilli,  in 
sputum,  in  the  experiments  of  Wyssokowitsch,  did  not  develop  tuber- 
culosis ;  and  in  guinea-pigs  the  smaller  the  number  injected  the  more 
protracted  the  course  of  the  disease  was  found  to  be. 

Tuberculosis  in  man  no  doubt  results,  in  a  large  proportion  of  the 
cases,  from  the  respiration,  by  a  susceptible  individual,  of  air  con- 
taining the  tubercle  bacillus  in  suspension  in  a  desiccated  condition. 
As  already  stated,  it  has  been  demonstrated  by  experiment  that  the 
bacillus  retains  its  vitality  in  desiccated  sputum  for  several  months. 
The  experiments  of  Cornet  have  demonstrated  that  in  the  dust  of 
apartments  occupied  by  tuberculous  patients  tubercle  bacilli  are  very 
commonly  present  in  sufficient  numbers  to  induce  tuberculosis  in 
guinea-pigs  inoculated  in  the  peritoneal  cavity  with  such  dust,  while 
negative  results  were  obtained  from  inoculations  with  dust  from 
other  localities.  In  view  of  these  facts  the  usual  mode  of  infection 
is  apparent.  Infection  may  also  occur  through  an  open  wound  or 
abrasion  of  the  skin,  as  in  the  small,  circumscribed  tumors  which 
sometimes  develop  upon  the  hands  of  pathologists  as  a  result  of 
handling  tuberculous  tissues.  A  few  instances  of  accidental  inocu- 
lation through  wounds  made  by  glass  or  earthen  vessels  containing 
tuberculous  sputum  have  also  been  recorded.  A  more  common  mode 
of  infection,  especially  in  children,  is  probably  by  way  of  the  intesti- 
nal glands,  from  the  ingestion  of  the  milk  of  tuberculous  cows.  That 
infection  may  occur  by  way  of  the  intestine  has  been  proved  by  ex- 
periments upon  rabbits,  which  develop  tuberculosis  when  fed  upon 
29 


410  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

tuberculous  sputum.  And  that  the  tubercle  bacillus  is  frequently,  if 
not  usually,  present  in  the  milk  of  tuberculous  cows  has  been  proved 
by  the  experiments  of  Bollinger,  Hirschberger,  Ernst,  and  others. 
In  Hirschberger's  investigations  milk  from  tuberculous  cows  induced 
tuberculosis  in  guinea-pigs,  when  injected  subcutaneously  or  into 
the  peritoneal  cavity,  in  fifty-five  per  cent  of  the  cases  studied 
(twenty).  The  conclusion  is  reached  that  the  milk  may  contain  tu- 
bercle bacilli  even  when  the  udder  of  the  cow  is  not  involved.  Ernst 
also,  from  an  examination  of  the  milk  from  thirty-six  tuberculous 
cows  in  which  the  udder  was  apparently  not  involved,  found  the 
tubercle  bacillus  by  microscopical  examination  in  five  per  cent  of  the 
samples  examined  (one  hundred  and  fourteen). 

The  prevalence  of  tuberculosis  among  cattle  is  shown  by  numer- 
ous investigations,  and  especially  by  the  official  inspections  of 
slaughtered  animals  made  in  Germany.  Thus  in  Saxony,  in  the 
year  1889,  of  611,511  cattle  examined  6,135  were  found  to  be  tubercu- 
lous (about  one  per  cent) ;  in  Berlin,  1887-1888,  out  of  130,733  ani- 
mals slaughtered  4,300  were  found  to  be  tuberculous  (3.2  per  cent). 
In  view  of  the  facts  stated  the  great  mortality  from  tubercular  dis- 
eases among  children,  many  of  whom  are  removed  from  other  prob- 
able sources  of  infection,  is  not  difficult  to  understand,  and  the 
practical  and  simple  method  of  preventing  infection  in  this  way,  af- 
forded by  the  sterilization  (by  heat)  of  milk  used  as  food  for  infants, 
must  commend  itself  to  all. 

54.    BACILLUS  TUBERCULOSIS   GALLINARUM. 

The  researches  of  Maffucci  (1889)  and  of  Cadiot,  Gilbert,  and 
Roger  (1890)  show  that  the  bacillus  obtained  from  spontaneous  tu- 
berculosis in  chickens,  although  closely  resembling  the  bacillus  of 
human  tuberculosis,  is  not  identical  with  it,  varying  especially  in  its 
pathogenic  power.  This  view  is  sustained  by  the  observations  of 
Koch,  who  says  in  his  address  before  the  Tenth  International  Medi- 
cal Congress  (Berlin,  1890) : 

"  The  care  which  it  is  necessary  to  exercise  in  judging  of  the  characters 
which  serve  to  differentiate  bacteria,  even  those  which  are  well  known,  I 
have  learned  in  the  case  of  the  tubercle  bacillus  This  species  is  so  definitely 
characterized  by  its  staining  reactions,  its  growth  in  pure  cultures,  and  its 
pathogenic  qualities,  and  indeed  by  each  of  these  characters,  that  it  seems 
iiiij).»>il>le  to  con  found  it  with  other  species.  Nevertheless  in  this  case  also 
one  should  not  rely  upon  a  single  one  of  the  characters  mentioned  for  de- 
termining the  species,  but  should  follow  the  safe  rule  that  all  available 
characters  should  he  considered,  and  the  identity  of  a  certain  bacterium 
should  only  be  regarded  as  demonstrated  when  it  has  been  shown  to  corre- 
spond in  all  of  these  particulars  When  I  made  my  first  researches  with 
reference  to  the  tubercle  bacillus  I  was  controlled  by  this  rule,  and  tested 
tubercle  bacilli  from  various  sources,  not  only  with  reference  to  their  stain- 
ing reactions,  but  also  with  reference  to  their  growth  in  culture  media  and 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  411 

pathogenic  characters.  Only  in  the  tuberculosis  of  chickens  I  was  not  able 
to  apply  this  rule,  as  at  that  time  it  was  not  possible  for  me  to  obtain  fresh 
material  from  which  to  make  pure  cultures.  As,  however,  all  other  forms 
of  tuberculosis  had  given  identical  bacilli,  and  the  bacilli  of  chicken  tuber- 
culosis in  their  appearance  and  behavior  towards  the  aniline  colors  entirely 
corresponded  with  these,  I  believed  myself  justified  in  assuming  their  iden- 
tity, notwithstanding  the  incompleteness  of  the  research.  Later  I  received 
pure  cultures  from  various  sources,  which  apparently  originated  from  tuber- 
cle bacilli,  but  in  several  regards  differed  from  these ;  especially  in  the  fact 
that  inoculation  experiments,  made  by  experienced  and  reliable  investigators, 
led  to  dissimilar  results,  which  it  was  necessary  to  regard  as  unexplained  con- 
tradictions. At  first  I  believed  that  these  differences  depended  upon  changes 
such  as  are  frequently  o  >served  in  pathogenic  bacteria,  when  these  are  culti- 
vated in  pure  cultures  outside  of  the  body  fora  long  time  under  more  or  less 
unfavorable  conditions.  In  order  to  solve  the  riddle  I  attempted  by  various 
influences  to  change  the  common  tubercle  bacilli  into  the  presumed  variety 
referred  to.  They  were  cultivated  for  several  months  at  so  high  a  tempera- 
ture that  only  a  scanty  growth  was  obtained;  in  other  experiments  still 
higher  temperatures  were  allowed  to  act  repeatedly  for  so  long  a  time  that 
the  cultures  were  brought  as  nearly  as  possible  to  the  point  of  killing  the 
bacilli.  In  a  similar  way  I  subjected  the  cultures  to  the  action  of  chemical 
agents,  of  light,  or  absence  of  moisture ;  they  were  cultivated  for  many  gen- 
erations in  association  with  other  bacteria ;  inoculated  successively  in  ani- 
mals having  but  a  slight  susceptibility.  But,  in  spite  of  all  these  attempts, 
only  slight  variations  were  obtained  in  their  characters — far  less  than  other 
pathogenic  bacteria  undergo  under  similar  circumstances.  Itappears,  there- 
fore, that  the  tubercle  bacilli  retain  their  characters  with  special  obstinacy ; 
this  is  in  accord  with  the  fact  that  pure  cultures  which  have  now  been  cul- 
tivated by  me  in  test  tubes  for  more  than  nine  years,  without  in  the  mean- 
time having  been  in  a  living  body,  are  still  entirely  unchanged  with  the  ex- 
ception of  a  slight  diminution  of  virulence.  ...  It  happened  about  a  year 
ago  that  I  received  a  living  chicken  which  was  suffering  from  tuberculosis, 
and  I  used  this  opportunity  to  make  cultures  directly  from  the  diseased  or- 
gans of  this  animal,  which  previously  I  had  not  been  able  to  do.  When  the 
cultures  grew  I  saw  to  my  surprise  that  they  had  precisely  the  appearance 
and  all  of  the  characters  possessed  by  the  enigmatical  cultures  resembling 
those  of  the  genuine  tubercle  bacillus.  Later  I  learned  that  these  also  ori- 
ginated from  tuberculosis  in  fowls,  but,  upon  the  assumption  that  all  forms 
of  tuberculosis  are  identical,  had  been  considered  genuine  tubercle  bacilli. 
A  verification  of  my  observations  I  find  in  the  recently  published  researches 
of  Prof.  Maffucci  with  reference  to  tuberculosis  of  fowls." 

According  to  Maffucci,  adult  chickens  are  refractory  against  the 
action  of  the  Bacillus  tuberculosis  from  man,  and  there  are  slight 
morphological  and  biological  differences  in  the  bacilli  from  the  two 
sources. 

Cadiot,  Gilbert,  and  Roger  (1891)  have  made  a  series  of  experi- 
ments with  the  bacillus  of  tuberculosis  in  fowls.  They  found 
the  bacilli  to  be  very  numerous  in  the  livers  of  chickens  suffering 
from  spontaneous  tuberculosis,  and  inoculated  with  material  from 
this  source  six  chickens,  five  rabbits,  and  twelve  guinea-pigs.  The 
chickens,  when  inoculated  in  the  cavity  of  the  abdomen  or  by  injec- 
tion into  a  vein,  died  in  from  forty-one  to  ninety-three  days  from 
general  tuberculosis.  Four  of  the  rabbits  died  of  general  tuberculosis, 
presenting  the  same  appearance  as  that  following  inoculation  with 


412  BACILLI  IN  CHRONIC   INFECTIOUS  DISEASES. 

bacilli  from  human  tuberculosis.  Of  the  guinea-pigs,  which  were 
inoculated  in  the  cavity  of  the  abdomen,  eleven  remained  in  good 
health  and  one  only  died  of  general  tuberculosis.  These  experi- 
ments show  a  decided  difference  in  the  pathogenic  properties  of 
tubercle  bacilli  from  the  two  sources,  for  the  guinea-pig  is  especially 
susceptible  to  tuberculosis  as  a  result  of  similar  inoculations  with 
bacilli  from  human  tuberculosis.  We  must  therefore  conclude  that 
the  bacillus  found  in  spontaneous  tuberculosis  in  fowls  is  a  distinct 
variety  of  Bacillus  tuberculosis.  Whether  this  variety  would  cause 
tuberculosis  in  man,  if  introduced  into  susceptible  subjects,  has  not 
been  determined ;  and,  as  pointed  out  by  Koch,  this  question  can 
only  be  answered  in  the  affirmative  if  it  should  be  obtained  in  pure 
cultures  from  cases  of  human  tuberculosis. 

Since  the  above  was  written  Maffucci  has  published  (1892)  an 
elaborate  memoir  upon  tuberculosis  of  fowls.  His  conclusions  are 
stated  as  follows : 

"  The  bacillus  of  tuberculosis  in  fowls  is  distinguished  from  that  of  tuber- 
culosis in  mammals  by  the  following  points  of  difference : 

**1.  It  does  not  induce  tuberculosis  in  guinea-pigs,  and  seldom  causes 
general  tuberculosis  in  rabbits. 

4  *  2.  The  cultures  in  various  media  have  a  different  appearance  from  those 
of  the  Bacillus  tuberculosis  of  mammals. 

"  3.  The  temperature  at  which  it  develops  varies  between  35°  and  45°  C., 
and  the  thermal  death-point  is  70°  C. 

"4.  At  45°  to  50°  C.  the  cultures  show  long,  thick,  and  branched  forms. 

"5.  The  bacillus  retains  its  vegetative  and  pathogenic  power  at  the  end 
of  two  years. 

"  6.  This  bacillus  produces  a  substance  which  is  toxic  for  guinea-pigs  and 
is  but  slightly  toxic  for  grown  fowls. 

"  7.  The  tuberculosis  produced  in  fowls  by  this  bacillus  is  without  giant 
cells." 

Additional  Notes  upon  the  Tubercle  Bacillus  (1895).— Several 
authors  (Metschnikoff,  Czaplewski,  Fischel)  have  described  branch- 
ing forms  of  the  tubercle  bacillus,  and  Lubinsky  (1895)  reports  that 
in  certain  media  it  grows  out  into  long  threads,  which,  however,  he 
has  never  observed  to  be  branched.  The  media  used  by  him  are  said 
to  give  a  more  abundant  growth  than  occurs  upon  glycerin-agar ; 
the  most  favorable  being  made  of  flesh-peptone  agar,  or  flesh -peptone 
bouillon,  containing  four  per  cent  of  glycerin  and  mashed  potato, 
one  kilo  of  finely  chopped  and  washed  potato  to  fifteen  hundred  cubic 
centimetres  of  water ;  this  is  cooked  for  three  or  four  hours  and  filtered 
—to  the  filtrate  is  added  four  per  cent  of  glycerin ;  one  and  a  half 
per  cent  of  agar  is  now  added  and  the  mixture  is  again  cooked  and 
filtered. 

Jones  (1895)  has  observed  the  branching  forms  previously  de- 
scribed by  several  authors,  and  states  that  they  are  only  found  upon 
the  surface  of  culture  media  where  there  is  free  access  of  oxygen. 


BACILLI   IN    CHRONIC    INFECTIOUS    DISEASES.  413 

He  concludes  that  the  tubercle  bacillus  does  not  form  endogenous 
spores,  such  as  are  found  in  various  other  bacilli,  but  that  in  the  rods 
and  branched  filaments  certain  objects  are  seen  which  are  probably 
reproductive  elements,  and  which  closely  resemble  similar  bodies 
("  Kolben  ")  seen  in  the  actinomyces  fungus,  to  which  Jones  believes 
the  tubercle  bacillus  is  closely  related. 

Prudden  and  Hodenpyl  (1891)  have  shown  that  the  injection  of 
dead  tubercle  bacilli  in  rabbits  gives  rise  to  the  development  of  nod- 
ules in  the  lung  containing  epithelioid  and  giant  cells,  but  that  these 
never  undergo  caseation.  This  fact  is  supposed  to  justify  the  infer- 
ence that  caseation  is  due  to  the  products  elaborated  during  the 
growth  of  living  tubercle  bacilli.  The  results  reported  by  Vissmann 
(1892)  correspond  with  those  reported  by  Prudden  and  Hodenpyl. 
Gamaleia  (1892)  has  also  obtained  nodules  with  epithelioid  and 
giant  cells  from  the  injection  of  dead  tubercle  bacilli,  but  in  his  ex- 
periments he  also  found  caseation  of  the  nodules.  Baumgarten  sug- 
gests that  this  was  probably  due  to  the  fact  that  there  were  some  liv- 
ing tubercle  bacilli  remaining  in  the  cultures  which  he  injected. 

Loomis  (1890)  and  Pizzini  (1892)  have  shown  that  living  tubercle 
bacilli  are  not  infrequently  found  in  the  bronchial  glands  of  individ- 
uals who  present  no  evidence  of  tubercular  disease  of  the  lungs  or  else- 
where. The  author  last  mentioned  inoculated  thirty  guinea-pigs 
with  the  bronchial,  mesenteric,  and  cervical  glands  of  thirty  in- 
dividuals in  whom  death  was  due  to  accident  or  acute  disease,  and 
who  were  free  from  tuberculosis.  Twelve  of  these  thirty  guinea- 
pigs  developed  tuberculosis  as  a  result  of  the  inoculation. 

Straus  (1894)  has  found  tubercle  bacilli  in  the  nasal  cavities  of 
healthy  individuals. 

Ernst  (1895),  as  the  result  of  extended  researches  made  under  the 
auspices  of  the  Massachusetts  Society  for  Promoting  Agriculture, 
has  arrived  at  the  following  conclusions  with  reference  to  the  pres- 
ence of  the  tubercle  bacillus  in  the  milk  of  tuberculous  cows : 

"  The  possibility  of  milk  from  tuberculous  udders  containing  the 
infectious  element  is  undeniable. 

"  With  the  evidence  here  presented,  it  is  equally  undeniable  that 
milk  from  diseased  cows  with  no  appreciable  lesion  of  the  udder  may, 
and  not  infrequently  does,  contain  the  bacillus  of  the  disease." 

De  Schweinitz  (1894)  has  found  that  by  continued  cultivation  in 
an  artificial  medium  the  tubercle  bacillus  becomes  attenuated,  so  that 
when  inoculated  into  guinea-pigs  these  animals  give  no  evidence  of 
tubercular  infection  for  six  months  or  more.  And  his  experiments 
indicate  that  animals  which  have  survived  an  inoculation  with  the 
attenuated  tubercle  bacillus  acquire  an  immunity  against  the  patho- 
genic action  of  virulent  cultures. 


414  BACILLI  IN   CHRONIC  INFECTIOUS  DISEASES. 

Amann  (1895)  has  given  in  the  Centralblatt  fur  Bakteriologie 
(Bd.  xvii.,  page  513)  a  detailed  account  of  his  method  for  demon- 
strating the  presence  of  tubercle  bacilli  in  sputum  by  sedimentation. 
He  mixes  the  sputum  with  two  to  four  volumes  of  cold  distilled 
water,  in  a  glass  cylinder  which  should  not  be  more  than  half  full. 
He  adds  one  cubic  centimetre  of  chloroform  and  a  small  quantity 
of  shot;  the  glass  cylinder  is  then  closed  with  a  rubber  cork  and  vio- 
lently shaken  for  some  minutes.  From  four  to  six  volumes  of  dis- 


Fio.  122.-Section  of  a  recent  lepra  nodule  of  the  Bkin.    X  950.    (Baumgarten.) 

tilled  water  are  then  added  and  the  mixture  is  placed  in  a  V-formed 
glass  tube  for  sedimentation ;  two  cubic  centimetres  of  carbol-f uchsin 
solution  are  added  and  distributed  by  gentle  agitation  of  the  tube. 
At  the  end  of  two  days  the  sedimentation  is  complete  and  the  stained 
bacilli,  cells,  connective-tissue  fibres,  etc.,  are  taken  up  with  a  pipette 
for  examination  under  the  microscope. 

55.    BACILLUS  LEPR^E. 

Discovered  by  Hansen  (1879),  chiefly  in  the  interior  of  the  peculiar 
round  or  oval  cells  found  in  leprous  tubercles.  Discovery  confirmed 
by  Neisser  (1879)  and  by  many  subsequent  observers. 

While  found  chiefly  in  the  leprous  tubercles  of  the  skin  and  mucous 
membranes,  the  bacilli  have  also  been  found  in  the  lymphatic  glands, 
the  liver,  the  spleen,  the  testicles,  and,  in  the  anesthetic  form  of  the 
<lisc;ise,  in  the  thickened  portions  of  nerves  involved  in  the  leprous 
process.  Some  observers  have  also  reported  finding  them  in  the 
blood,  but  this  appears  to  be  quite  exceptional.  In  the  leprous  cells 
they  are  commonly  found  in  great  numbers,  and  they  may  also  be 
seen  in  the  lymph  spaces  outside  of  these  cells.  They  are  not  found 
in  the  epidermal  layer  of  the  skin,  but,  according  to  Babes,  they  may 
penetrate  the  hair  follicles. 

Morphology. — The  bacillus  of  leprosy  resembles  the  tubercle  ba- 
cillus in  form,  but  is  of  more  uniform  length  and  not  so  frequently 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  415 

bent  or  curved.  The  rods  have  pointed  ends ;  and  in  stained  pre- 
parations unstained  spaces,  similar  to  those  observed  in  the  tubercle 
bacillus  and  generally  assumed  to  be  spores,  are  to  be  seen,  although 
not  quite  so  distinctly  as  in  the  latter.  The  bacilli  are  said  by  Fliigge 
to  be  from  four  to  six  /*  in  length  and  less  than  one  /*  in  width — 
probably  considerably  less,  for  the  same  author  states  that  the  tubercle 
bacillus  has  about  the  diameter  of  the  bacillus  of  mouse  septicaemia, 
and  this  is  given  as  0.2  P-. 

This  bacillus  stains  readily  with  the  aniline  colors  and  also 
by  Gram's  method.  Although  it  differs  from  the  tubercle  bacillus 
in  the  ease  with  which  it  takes  up  the  ordinary  aniline  colors,  it  re- 
sembles it  in  retaining  its  color  when  subsequently  treated  with 
strong  solutions  of  the  mineral  acids.  Double-stained  prepara- 
tions are  therefore  easily  made  by  first  staining  sections  or  cover- 
glass  preparations  in  Ziehl's  carbol-f  uchsin  solution  or  in  an  aqueous 
solution  of  methyl  violet,  decolorizing  in  acid,  washing  in  alcohol, 
and  counter-staining  with  methylene  blue — or,  if  methyl  violet  was 
used  in  the  first  instance,  with  vesuvin. 

Biological  Characters. — The  earlier  attempts  to  cultivate  this 
bacillus  were  without  success,  but  recently  Bordoni-Uffreduzzi  has 
obtained  from  the  marrow  of  the  bones  of  a  leper  a  bacillus  which 
he  believes  to  be  the  leprosy  bacillus,  and  which  he  was  able  to  culti- 
vate upon  blood  serum  to  which  a  certain  amount  of  peptone  and  of 
glycerin  had  been  added.  At  first  this  bacillus  only  grew  with  diffi- 
culty and  in  the  incubating  oven  ;  but  after  it  had  been  cultivated 
artificially  through  a  number  of  generations  it  is  said  to  have  grown 
upon  ordinary  nutrient  gelatin  at  the  room  temperature.  The  bacillus 
obtained  in  this  way  is  said  to  have  retained  its  color  when  treated 
with  acids,  after  having  been  stained  with  aniline-f uchsin,  correspond- 
ing in  this  respect  with  the  bacillus  of  leprosy  and  the  tubercle  ba- 
cillus. But  it  differed  considerably  in  its  morphology  from  the  Ba- 
cillus leprsBas  seen  in  the  tissues  of  lepers,  being  considerably  thicker, 
and  it  was  not  so  promptly  stained  by  the  aniline  colors  as  is  the 
bacillus  found  in  the  tissues.  Moreover,  attempts  to  cultivate  the 
same  bacillus  from  leprous  tubercles  of  the  skin  were  unsuccessful, 
as  were  also  inoculation  experiments  into  the  anterior  chamber  of  the 
eye  in  rabbits.  It  is  therefore  a  matter  of  doubt  as  to  whether  the 
bacillus  obtained  by  Bordoni-Uffreduzzi  is  identical  with  that  present 
in  such  numbers  in  the  cells  of  the  leprous  tubercles,  to  which  the 
name  Bacillus  leprse  has  been  given. 

Some  of  the  earlier  observers  described  the  bacillus  of  leprosy  as 
motile,  but  this  assertion  seems  to  have  been  based  upon  some  error 
of  observation,  and  it  is  now  generally  agreed  that,  like  the  tubercle 
bacillus,  it  is  without  proper  movements.  The  question  of  spore  for- 


416  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

mation  has  not  been  definitely  settled.  As  before  remarked,  un- 
stained portions,  occurring  at  regular  intervals,  are  seen  in  the  rods  in 
stained  preparations  ;  but  no  satisfactory  evidence  has  been  presented 
to  show  that  these  are  truly  reproductive  spores. 

Pathogenesis. — The  inference  that  the  bacillus  above  described 
bears  an  etiological  relation  to  the  disease  with  which  it  is  associated 
is  based  upon  the  demonstration  of  its  constant  presence  in  leprous 
tissues — which  has  now  been  repeatedly  made  in  various  and  distant 
parts  of  the  world — and  of  its  absence  from  the  same  tissues  involved 
in  different  morbid  processes.  As  it  has  not  been  obtained  in  pure 
cultures,  the  final  proof  of  such  etiological  relation  is  still  wanting. 
We  have,  however,  experimental  evidence  to  show  that  leprous  tis- 
sues containing  this  bacillus  are  infectious  and  may  reproduce  the 
disease.  The  experiment  has  been  made  upon  man  by  Arning,  who 
inoculated  a  condemned  criminal  subcutaneously  with  fresh  leprous 
tubercles.  The  experiment  was  made  in  the  Sandwich  Islands,  and 
the  man  was  under  observation  until  his  death  occurred  from  leprosy 
at  the  end  of  about  five  years.  The  first  manifestations  of  the  disease 
became  visible  in  the  vicinity  of  the  point  of  inoculation  several 
months  after  the  experimental  introduction  of  the  infectious  material. 

Positive  results  have  also  been  reported  in  the  lower  animals  by 
Damsch,  by  Vossius,  and  by  Melcher  and  Ortmann.  The  last-named 
investigators  inoculated  rabbits  in  the  anterior  chamber  of  the  eye 
with  portions  of  leprous  tubercles  excised  for  the  purpose  from  a 
leper.  The  animals  died  from  general  infection  at  the  end  of  several 
months,  and  the  characteristic  tubercles  containing  the  bacillus  were 
distributed  through  the  various  organs. 

Wolters  (1893)  who  has  made  numerous  inoculation  experiments 
and  has  made  a  critical  review  of  all  the  recorded  experimental  evi- 
dence, arrives  at  the  conclusion  that  the  comparatively  small  number 
of  successful  results  reported  cannot  be  accepted  as  evidence  that 
leprosy  can  be  transmitted  to  the  lower  animals  by  inoculation.  He 
believes  that  in  some  cases  the  tubercle  bacillus  has  been  present  in 
the  material  inoculated  and  that  the  infectious  process  following  the 
inoculation  was  tuberculous  and  not  leprous.  In  inoculations  into 
the  anterior  chamber,  in  the  eyes  of  rabbits,  the  considerable  number 
of  bacilli  introduced  with  the  leprous  tissue  remain  and  retain  their 
staining  properties,  so  that  the  bacilli  originally  introduced  are  found 
in  the  leucocytes  of  the  inflammatory  exudate  or  granulation  tissue 
formed  as  a  result  of  the  introduction  of  foreign  material.  Wolters 
also  doubts  whether  the  few  successful  results  reported  in  the  culti- 
vation of  the  lepra  bacillus  are  trustworthy.  He  has  never  succeeded 
in  his  efforts  to  cultivate  the  bacillus. 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


417 


56.    BACILLUS  MALLEI. 

Synonyms.— The  bacillus  of  glanders;  Der  Rotzbacillus,  Ger.; 
Bacille  de  la  morve,  Fr. 

Discovered  by  Loffler  and  Schutz  (1882),  and  proved  to  be  the 
cause  of  glanders  by  the  successful  inoculation  of  pure  cultures. 
Found  especially  in  the  recent  nodules  in  animals  infected  with 
glanders  ;  also  in  the  same  after  ulceration,  and  in  the  discharge 
from  the  nostrils,  pus  from  the  specific  ulcers,  etc. ;  sometimes  in  the 
blood  of  infected  animals  (Weichselbaum). 

Morphology. — Bacilli  with  rounded  ends,  straight  or  slightly 
curved,  rather  shorter  and  decidedly  thicker  than  the  tubercle  bacil- 
lus ;  usually  solitary,  but  occasionally  united  in 
pairs,  or  in  filaments  containing  several  elements 
(in  potato  cultures).  In  stained  preparations 
unstained  or  feebly  stained  spaces  are  seen  in 
the  rods,  alternating  with  the  deeply  stained 
protoplasm  of  the  cell.  As  in  the  tubercle  bacil- 
lus, which  presents  a  similar  appearance,  these 
spaces  have  been  supposed  by  some  bacteriolo- 
gists to  represent  spores  ;  but  Loffler  believes 
them  to  represent  rather  a  degeneration  of  the 
protoplasm.  Baumgarten  and  Rosenthal  claim 
to  have  demonstrated  the  presence  of  spores  by  the  use  of  Neisser's 
method  of  staining,  but  they  do  not  consider  it  established  that  the 
unstained  spaces  in  the  rods  referred  to  are  of  this  nature. 

The  glanders  bacillus  may  be  stained  with  aqueous  solutions  of 
the  aniline  colors,  but  the  staining  is  more  intense  when  the  solution 


FIG.  123.— Bacillus  mal- 
lei, x  1,000.  From  a  pho- 
tomicrograph. CFriinkel 
and  Pfeiffer.) 


Fia.  124.— Section  of  a  glanders  nodule.    X  700.    (Flugge.) 

is  made  feebly  alkaline.  Add  to  three  cubic  centimetres  of  a  1 : 10,000 
solution  of  caustic  potash,  in  a  watch  glass,  one  cubic  centimetre  of 
a  saturated  alcoholic  solution  of  an  aniline  color  (methylene  blue, 


418  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

gentian  violet  or  fuchsin);  or  the  aniline-water-fuchsin,  or  methyl 
violet  solution  of  Ehrlich  may  be  used,  with  the  addition  just  be- 
fore use  of  an  equal  quantity  of  1 : 10,000  solution  of  caustic  potash. 
Loftier  recommends  that  cover-glass  preparations  be  placed  in  Ehr- 
lich 's  solution  and  heated  for  five  minutes;  then  decolorized  in  a  one- 
per-cent  solution  of  acetic  acid  to  which  sufficient  tropseolin  has 
been  added  to  give  it  the  yellow  color  of  Rhine  wine  ;  then  quickly 
washed  in  distilled  water.  This  bacillus  presents  the  peculiarity  of 
losing  very  quickly  in  decolorizing  solutions  the  color  imparted  to  it 
by  the  aniline  staining  solutions.  For  this  reason  the  staining  of  the 
bacillus  in  sections  is  attended  with  some  difficulty.  Loffler  recom- 
mends his  alkaline  methylene-blue  solution  for  staining  sections  ;  and 
for  decolorizing,  a  mixture  containing  ten  cubic  centimetres  of  distilled 
water,  two  drops  of  strong  sulphuric  acid,  and  one  drop  of  a  five- 
per-cent  solution  of  oxalic  acid.  Thin  sections  should  be  left  in  this 
acid  solution  about  five  seconds.  The  method  more  recently  recom- 
mended by  Kuhne  also  gives  good  results  in  skilful  hands  (see  p.  34). 
Biological  Characters. — An  aerobic,  non-motile,  parasitic 
bacillus,  which  may  be  cultivated  in  various  artificial  media  at  a 
temperature  of  37°  C.  The  lowest  temperature  at  which  develop- 
ment occurs  (22°  C. — Loffler)  is  a  little  above  that  at  which  nutrient 
gelatin  is  liquefied  ;  the  highest  limit  is  43°  C.  According  to  Frankel, 
the  glanders  bacillus  is  a  facultative  anaerobic.  Baumgarten  and 
Rosenthal  claim  to  have  demonstrated  the  presence  of  spores  by 
Neisser's  method  of  staining.  Loffler  was  led  to  doubt  the  forma- 
tion of  spores  from  the  results  of  his  experiments  upon  the  thermal 
death-point  of  this  bacillus,  and  its  comparatively  slight  resistance 
to  desiccation  and  destructive  chemical  agents.  He  found  that  ex- 
posure for  ten  minutes  to  a  temperature  of  55°  C.,  or  for  five  minutes 
to  a  three-  to  five-per-cent  solution  of  carbolic  acid,  or  for  two  min- 
utes to  a  1  :  5,000  solution  of  mercuric  chloride,  was  effectual  in  de- 
stroying its  vitality.  As  a  rule,  the  bacilli  do  not  grow  after  having 
been  preserved  in  a  desiccated  condition  for  a  few  weeks  ;  and  in  a 
moist  condition  the  cultures  cannot  be  preserved  longer  than  three 
or  four  months— usually  not  so  long  as  this  (Loffler).  The  bacillus 
does  not  grow  in  infusions  of  hay,  straw,  or  horse  manure,  and  it  is 
doubtful  whether  it  finds  conditions  in  nature  favorable  for  its  sap- 
rophytic  existence.  It  grows,  in  the  incubating  oven,  in  neutral 
bouillon,  in  nutrient  gelatin,  or  in  nutrient  agar,  and  still  better  in 
glycerin-agar.  Upon  the  last-mentioned  medium  it  grows,  even  at 
the  room  temperature  (Kranzfeld),  but  better  still  in  the  incubating 
oven,  as  a  pale- white,  transparent  streak  along  the  line  of  inocula- 
tion, which  at  the  end  of  six  or  seven  days  may  have  a  width  of 
seven  to  eight  millimetres.  According  to  Raskina,  nutrient  agar 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  419 

made  with  milk  forms  an  extremely  favorable  medium,  upon  which 
a  thick,  pale- white  layer  develops  in  two  or  three  days,  which  on  the 
third  or  fourth  day  acquires  an  amber-yellow  color,  and  the  deeper 
layers  acquire  a  brownish-red  tint. 

The  growth  upon  solidified  blood  serum,  in  the  course  of  three  or 
four  days  at  37°  C.,  consists  of  yellowish,  transparent  drops,  which 
later  coalesce  into  a  viscid  layer,  which  has  a  milky  appearance  from 
the  presence  of  numerous  small  crystals  (Baumgarten).  The  growth 
upon  cooked  potato  is  especially  characteristic.  In  the  incubating 
oven,  at  the  end  of  two  or  three  days,  a  rather  thin,  yellowish,  trans- 
parent layer  develops,  which  resembles  a  thin  layer  of  honey.  Later 
this  ceases  to  be  transparent,  and  the  amber  color  changes,  at  the 
end  of  six  to  eight  days,  to  a  reddish-brown  color ;  and  outside  of 
the  reddish-brown  layer,  with  more  or  less  irregular  outlines,  the 
potato  for  a  short  distance  acquires  a  greenish-yellow  tint. 

Pathogenesis. — Glanders  occurs  principally  among  horses  and 
asses,  but  may  be  contracted  by  man  from  contact  with  infected 
animals ;  it  has  also  been  communicated,  in  one  instance  with  a  fatal 
result,  by  subcutaneous  inoculation,  resulting  accidentally  from  the 
use  of  an  imperfectly  sterilized  hypodermic  syringe  which  had  pre- 
viously been  used  for  injecting  cultures  of  the  bacillus  into  guinea- 
pigs.  The  field  mouse  and  the  guinea-pig  are  especially  susceptible 
to  infection  by  experimental  inoculations  ;  the  cat  and  the  goat  may 
be  infected  in  the  same  way.  Lions  and  tigers  in  menageries  are 
said  to  have  contracted  glanders  from  being  fed  upon  the  flesh  of  in- 
fected animals  (Baumgarten).  Rabbits  have  but  slight  susceptibility, 
and  the  same  is  true  of  sheep  and  dogs ;  swine,  cattle,  white  mice, 
and  common  house  mice  are  immune. 

The  etiological  relation  of  the  bacillus  is  fully  established  by  the 
experiments  of  Loffler  and  Schutz,  confirmed  by  other  bacteriologists, 
which  show  that  pure  cultures  injected  into  horses,  asses,  and  other 
susceptible  animals,  produce  genuine  glanders.  The  disease  is  char- 
acterized in  the  equine  genus  by  the  formation  of  ulcers  upon  the 
nasal  mucous  membrane,  which  have  irregular,  thickened  margins 
and  secrete  a  thin,  virulent  mucus ;  the  submaxillary  lymphatic 
glands  become  enlarged  and  form  a  tumor  which  is  often  lobulated  ; 
other  lymphatic  glands  become  inflamed,  and  some  of  them  suppurate 
and  open  externally,  leaving  deep,  open  ulcers ;  the  lungs  are  also 
involved  and  the  breathing  becomes  hurried  and  irregular.  In  farcy, 
which  is  a  more  chronic  form  of  the  same  disease,  circumscribed 
swellings,  varying  in  size  from  a  pea  to  a  hazelnut,  appear  on  differ- 
ent parts  of  the  body,  especially  where  the  skin  is  thinnest ;  these 
suppurate  and  leave  angry-looking  ulcers  with  ragged  edges,  from 
which  there  is  an  abundant  purulent  discharge.  The  specific  bacillus 


420 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


can  easily  be  obtained  in  pure  cultures  from  the  interior  of  suppurat- 
ing nodules  and  glands  which  have  not  yet  opened  to  the  surface, 
and  the  same  material  will  give  successful  results  when  inoculated 
into  susceptible  animals.  But  the  discharge  from  the  nostrils  or  from 
an  open  ulcer  contains  comparatively  few  bacilli ;  and  as  these  are 
associated  with  various  other  bacteria  which  grow  more  readily  in 
our  culture  media,  it  is  not  easy  to  obtain  pure  cultures,  by  the  plate 
method,  from  such  material. 

In  the  guinea-pig  subcutaneous  inoculation  is  followed  in  four  or 
five  days  by  tumefaction  at  the  point  of  inoculation,  and  after  a  time 
;i  prominent  tumor  with  caseous  contents  is  developed  ;  ulceration  of 
the  skin  follows,  and  a  chronic,  purulent  ulcer  with  irregular,  indu- 
rated margins  results ;  after  a  time  this  may  cicatrize.  Meanwhile 
the  lymphatic  glands  become  involved,  and  the  symptoms  of  general 


Fio.  126.— Section  through  a  glanders  nodule  in  liver  of  field  mouse.    Tissue  x  250.    Bacilli 
X  600.    (Baumsarten.) 

infection  are  developed  at  the  end  of  four  or  five  weeks ;  the  glands 
suppurate,  and  in  males  the  testicles  are  also  involved ;  finally  a  dif- 
fuse inflammation  of  the  joints  occurs,  and  death  results  from  ex- 
haustion. In  the  guinea-pig  the  specific  ulcers  upon  the  nasal  mu- 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  421 

cous  membrane,  which  characterize  the  disease  in  the  horse,  are  rarely 
developed  to  any  great  extent. 

In  field  mice  general  infection  occurs  at  once  as  a  result  of  the 
subcutaneous  injection  of  a  small  quantity  of  a  pure  culture,  and  the 
animal  dies  at  the  end  of  three  or  four  days.  Upon  post-mortem 
examination  the  principal  changes  are  found  in  the  liver  and  in  the 
greatly  enlarged  spleen.  Scattered  through  these  organs  are  minute 
gray  points  which  are  scarcely  visible  to  the  naked  eye.  In  the 
guinea-pig,  which  succumbs  at  a  later  date,  these  nodules  are  larger 
and  closely  resemble  miliary  tubercles,  both  macroscopically  and 
under  the  microscope,  in  stained  sections  of  the  tissues.  Similar 
nodules  are  also  found  in  the  kidneys  and  in  the  lungs ;  they  have  a 
decided  tendency  to  undergo  purulent  degeneration.  The  bacilli  are 
found  principally  in  these  nodules,  of  recent  formation,  and  are  com- 
monly associated  in  groups,  as  if  they  had  been  enclosed  in  the  inte- 
rior of  a  cell  the  membranous  envelope  of  which  had  undergone 
degeneration  and  disappeared. 

As  before  remarked,  it  is  not  an  easy  matter  to  demonstrate  the 
bacillus  in  sections  of  the  tissues  containing  these  nodules,  owing  to 
the  facility  with  which  they  lose  their  color  in  alcohol  and  other  de- 
colorizing agents.  For  this  reason  it  will  be  best  to  dehydrate  sec- 
tions by  the  use  of  aniline  oil  (Weigert's  method)  or  to  resort  to 
Kiihne's  method  of  staining.  • 

It  is  also  difficult  to  demonstrate  the  presence  of  the  bacillus  in 
nodules  which  have  undergone  purulent  degeneration,  in  the  secre- 
tions from  the  nostrils  of  horses  suffering  from  glanders,  or  in  the 
pus  from  the  specific  ulcers  and  suppurating  glands  ;  for  they  are 
present  in  comparatively  small  numbers.  But  the  virulent  nature  of 
these  discharges  is  shown  by  inoculations  into  guinea-pigs  or  mice, 
and  it  is  easier  to  obtain  a  pure  culture  from  such  virulent  material 
by  first  inoculating  a  susceptible  animal  than  directly  by  the  plate 
method;  for  the  small  number  of  bacilli  present,  and  their  associa- 
tion with  other  bacteria  which  develop  more  rapidly  in  our  culture 
media,  make  this  a  very  uncertain  procedure.  For  establishing  the 
diagnosis  of  glanders,  therefore,  Loffler  recommends  the  inoculation 
of  guinea-pigs  with  pus  from  a  suppurating  gland  or  ulcer,  or  the 
nasal  discharge  from  a  suspected  animal,  rather  than  a  direct  attempt 
to  demonstrate  the  presence  of  the  bacillus  by  staining  and  culture 
methods. 

The  method  proposed  by  Strauss  gives  more  prompt  results. 
This  consists  in  the  intraperitoneal  injection  of  cultures  or  of  the 
suspected  products  into  the  cavity  of  the  abdomen  of  male  guinea- 
pigs.  If  the  glanders  bacillus  is  present  the  diagnosis  may  be  made 
within  three  or  four  days  from  the  infectious  process  established  in 


422  BACILLI   IN   CHRONIC   INFECTIOUS  DISEASES. 

the  testicles.  At  the  end  of  this  time  the  scrotum  is  red  and  shining, 
the  epidermis  desquamates,  and  suppuration  occurs,  the  pus  some- 
times perforating  the  integument.  This  pus  is  found  to  contain  the 
glanders  bacillus.  The  animal  usually  dies  in  the  course  of  twelve 
to  fifteen  days.  When  the  animals  are  killed  three  or  four  days 
after  the  inoculation,  the  two  layers  of  the  tunica  vaginalis  testis 
are  found  to  be  covered  with  a  purulent  exudate  containing  the 
glanders  bacillus  and  to  be  more  or  less  adherent.  Even  as  early 
as  the  second  day  the  tunica  vaginalis  is  seen  to  be  covered  with 
granulations. 

An  attenuation  of  virulence  occurs  in  cultures  whicn  have  been 
kept  for  some  time,  and  inoculations  with  such  cultures  may  give  a 
negative  result ;  or,  when  considerable  quantities  are  injected,  may 
produce  a  fatal  result  at  a  later  date  than  is  usual  when  small 
amounts  of  a  recent  culture  are  injected  into  susceptible  animals. 

Kalning,  Preusse,  and  Pearson  have  obtained  from  cultures  of 
the  glanders  bacillus  a  glycerin  extract  similar  to  the  crude  tubercu- 
lin of  Koch — mallein.  This,  when  injected  into  animals  suffering 
from  glanders,  gives  rise  to  a  considerable  elevation  of  temperature, 
and  it  is  used  as  a  means  of  diagnosis  in  cases  of  suspected  infection  in 
animals  in  which  the  usual  symptoms  have  not  yet  manifested  them- 
selves. The  value  of  the  test  has  been  demonstrated  by  numerous 
experiments. 

Bonome  (1894),  as  a  result  of  extended  researches,  arrives  at  the 
following  conclusions : 

"1.  The  bacillus  is  found  not  only  in  the  diseased  tissues  and 
purulent  discharges,  but  also  in  the  urine  and  milk  of  infected  ani- 
mals. 

"  2.  The  bacillus  is  found  in  the  foetus  of  infected  animals  even 
when  the  placenta  is  free  from  any  pathological  change. 

"  3.  The  glanders  bacillus  is  very  sensitive  to  desiccation  and  will 
not  grow  after  being  preserved  for  ten  days  at  25°  C. 

"  4.  In  distilled  water  the  bacillus  dies  out  in  six  days. 

"5.  On  the  contrary,  when  protected  from  desiccation  it  resists 
a  comparatively  high  temperature — 70°  C.  for  six  hours;  a  temper- 
ature of  90°  to  100°  C.  destroys  it  in  three  minutes." 

57.    BACILLUS  OP    LUSTGARTEN. 

%//»// .//w.—  Sypliillis  bacillus. 

,...  Found  by  Lustgarten  (1884)  in  syphilitic  lesions  and  secretions  of  syphi- 

5  ulcers  and  believed  by  him  to  be  the  specific  infectious  agent  in  this 

Jease.     JNo  satisfactory  experimental  evidence  that  this  is  the  case  has  yet 

been  obtained. 

.1A,/V,/,, ,/,,,,/,.     Straigbt  or  curved  bacilli,  whirl,   bear  considerable  resem- 
blance to  tubercle  bacilli,  but  differ  from  them  in  the  staining  reactions, 
usually  more  or  less  curved,  or  bent  at  a  sharp  angle,  or  S-shaped  ; 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  423 

the  ends  often  present  slight  knob -like  swellings  ;  the  length  is  from  three 
and  one-half  p  to  four  and  one-half  /",  and  the  diameter  is  from  0.25  to  0.3  p. 
With  a  high  power  the  contour  is  seen  to  be  not  quite  regular,  but  wavy  in 
outline,  and  bright  shining  spaces  in  the  deeply  stained  rods  may  be  ob- 
served ;  these,  from  two  to  four  in  a  single  rod,  are  believed  by  Lustgarten 
to  be  spores.  The  bacilli  are  not  found  free  in  the  tissues,  but  are  enclosed 
in  cells  of  a  round-oval  or  polygonal  form,  which  are  said  to  be  about  double 
the  size  of  a  white  blood  corpuscle.  The  bacilli  are  not  numerous,  and  very 
commonly  only  one  or  two  are  found  in  a  single  cell,  but  groups  of  six  or 
eight  may  sometimes  be  seen,  especially  upon  the  margins  of  a  syphilitic 
lesion,  and  in  the  tissues  in  the  immediate  vicinity  of  the  infiltration,  which 
show  but  little  change  or  are  apparently  healthy  (Lustgarten). 

The  presence  of  these  bacilli  in  syphilitic  lesions  was  demonstrated  by 
Lustgarten  by  the  following  staining  method :  The  thin  sections  are  placed 
in  the  Ehrlich-Weigert  gentian- violet  solution  (one  hundred  parts  aniline 
water,  eleven  parts  saturated  alcoholic  solution  of  gentian  violet)  for  from 
twelve  to  twenty  four  hours  at  the  room  temperature,  and  two  hours  in  the 
incubating  oven  at  40°  C.  The  sections  are  then  thoroughly  washed  in  alco- 
hol and  placed  for  ten  seconds  in  a  1.5-per-cent  solution  of  potassium  per- 
manganate; in  this  solution  a  precipitate  of  peroxide  of  manganese  is 


FIQ.  126.  FIG.  127. 

FIG.  126.— Migrating  cell  containing  syphilis  bacilli.    (Lustgarten. ) 
FIG.  127  —Pus  from  hard  chancre  containing  syphilis  bacilli.     (Lustgarten.? 

formed,  which  adheres  to  the  section  ;  this  is  dissolved  and  washed  off  in  a 
dilute  aqueous  solution  of  pure  sulphuric  acid;  the  sections  are  then  washed 
in  water,  and,  if  not  completely  decolorized,  are  returned  for  a  few  seconds  to 
the  permanganate  solution  and  again  washed  off  in  the  acid;  it  may  be 
necessary  to  repeat  this  operation  three  or  four  times.  Finally  the  sections 
are  dehydrated  and  mounted  in  balsam  in  the  usual  manner.  Cover-glass 
preparations  are  made  in  the  same  way,  except  that,  after  being  taken  from 
the  staining  solution,  they  are  washed  off  in  water  instead  of  in  alcohol. 

Another  method  of  staining,  recommended  by  De  Giacorna,  consists  in 
placing  the  sections  for  twenty-four  hours  in  aniline-water-fuchsin  solution 
(cover-glass  preparations  may  be  stained  in  the  same  solution,  hot,  in  a  few 
minutes),  then  washing  them  in  water,  and  decolorizing  in  a  solution  of  per- 
chloride  of  iron— first  in  a  dilute  and  then  in  a  saturated  solution. 

The  method  of  staining  employed  by  Lustgarten  serves  to  differentiate 
his  bacillus  from  many  other  microorganisms,  but  not  from  the  tubercle  ba- 
cillus and  the  bacillus  of  leprosy,  which,  as  he  pointed  out,  may  be  stained 
in  the  same  way.  And  it  has  since  been  shown  by  Alvarez  and  Tavel,  and 
by  Matterstock,  that  in  smegma  from  the  prepuce  or  the  ^  vulva,  bacilli  are 
found  which  have  the  same  staining  reaction  and  are  similar  in  their  mor- 
phology to  the  bacillus  of  Lustgarten.  This  by  no  means  proves  that  the 


424  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

smegma  bacilli  found  under  the  prepuce  of  healthy  persons  are  identical 
•with  the  bacilli  found  by  Lustgarten  and  others  in  sections  of  tissues  involved 
in  syphilomata.  In  the  absence  of  pure  cultures  and  inoculation  experiments 
it  is  impossible  to  establish  identity,  however  similar  may  be  the  characters 
referreu  to.  Several  well-known  pathogenic  bacilli  resemble  quite  as  closely 
in  these  particulars  other  bacilli  which  have,  nevertheless,  been  differentiated 
from  them  by  culture  and  inoculation  experiments.  We  may  mention 
especially  in  this  connection  the  bacillus  of  diphtheria,  as  obtained  from  the 
pseudo-membranous  exudation  in  a  genuine  case  of  this  disease,  and  the 
pseudo  diphtheria  bacilli  found  by  Roux  and  Yersin  in  the  fauces  of  healthy 
children.  On  the  other  hand,  since  it  has  been  shown  that  similar  bacilli 
are  common  in  preputial  smegma,  we  cannot  attach  great  importance  to  the 
finding  of  Lustgarten's  bacillus  in  primary  syphilitic  sores ;  and  it  has  not 
been  found  in  sufficient  numbers,  or  with  sufficient  constancy,  by  those  who 
have  searched  for  it  subsequently  to  the  publication  of  Lustgarten's  inves- 
tigations, to  give  strong  support  to  the  view  that  it  is  the  specific  infectious 
agent  in  syphilis.  Baumgarten,  who  has  searched  in  vain  for  Lustgarten's 
bacillus  in  uncomplicated  visceral  syphilomata,  suggests  that  the  bacilli 
found  occasionally  in  such  lesions  were  perhaps  tubercle  bacilli  and  repre- 
sented a  mixed  infection.  As  the  bacillus  under  consideration  has  not  been 
obtained  in  cultures,  we  have  no  information  as  to  its  biological  characters 
and  pathogenesis. 

THE  SYPHILIS  BACILLUS  OF  EVE  AND  LINGARD. 
Eve  and  Lingard  (1886)  report  that  they  have  obtained  in  cultures  from 
the  blood  and  diseased  tissues  of  syphilitics  who  have  not  undergone  mer- 
curial treatment,  bacilli  which  in  their  form  and  dimensions  resemble  the 
tubercle  bacilli,  but  which  stain  readily  by  the  common  aniline  colors  and 
by  Gram's  method,  and  are  not  stained  by  Lustgarten's  method.  They  grow 
readily  upon  solidified  blood  serum,  forming  a  thin,  pale-yellow  or  brown- 
ish-yellow layer.  Inoculations  of  pure  cultures  into  apes  were  without 
result.  The  negative  results  which  have  attended  the  culture  experiments 
and  microscopical  examinations  of  the  blood  and  diseased  tissues,  made  by 
many  competent  bacteriologists  in  other  parts  of  Europe,  make  it  appear 
probable  that  the  bacilli  described  by  the  English  investigators  named  belong 
to  some  saprophytic  species,  and  that  they  are  not  usually  present  in  syphilo- 
mata or  the  blood  of  syphilitic  patients. 

MICROCOCCI  OF  DISSE  AND   TAGUCHI. 

Disse  and  Taguchi  (1886)  claim  to  have  obtained  from  the  blood  of  syphi- 
litics micrococci  which  they  were  able  to  cultivate  in  artificial  media  at  20 
to  40°  C.,  and  which  formed  on  the  surface  of  such  media  a  grayish- white 
layer  consisting  of  diplococci  which  are  motile  and  of  larger  motion  less  cocci. 
The  diplococci  are  said  to  originate  from  division  of  the  larger  cocci.  Inocu- 
lations into  rabbits,  dogs,  and  sheep  gave  rise  to  chronic  interstitial  inflam- 
matory processes  in  the  lungs  and  liver,  to  granulomata  in  various  organs, 
and  to  fattv  degenerative  changes  in  the  walls  of  the  arteries,  which,  in  the 
opinion  of  the  authors  named,  correspond  with  the  pathological  changes 
produced  by  syphilitic  infection  in  man.  We  remark,  with  reference  to  the 
supposed  etiological  relation  of  this  coccus,  that  bacteriologists  in  Europe 
have  not  confirmed  the  authors  named  as  to  the  presence  of  this  micrococcus 
in  the  blood  of  syphilitics,  and  that  the  micrococcus  of  progress! vegranuloma 
formation  described  bv  Manfredi  produces  similar  pathological  changes  in 
inoculated  animals;  also  that  there  is  no  evidence  that  the  animals  experi- 
mented upon  are  subject  to  syphilitic  infection. 

BACILLUS   OF   GOLASZ. 

Golasz  (1894)  has  published  in  the  Comptes  Rendus  of  the  French  Acad- 
eim  a  inscription  of  a  "polymorphous  microbe,"  which  he  claims  to  have 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


425 


first  discovered  in  1888  in  syphilitic  vegetations,  and  subsequently  to  have 
cultivated  from  the  blood  of  syphilitic  patients,  in  a  culture  medium  consist- 
ing of  nuclein  from  the  spleen  of  persons  free  from  syphilitic  infection. 

58.    BACILLUS   OF  RHINOSCLEROMA. 

First  observed  by  Von  Frisch  (1882)  in  the  newly  formed  tubercles  of 
rhinoscleroma.  Cultivated  by  Paltauf  arid  Von  Eiselberg  (1880). 

Rhinoscleroma  is  a  chronic  affection  of  the  skin,  and  especially  of  the 
mucous  membrane  of  the  nares,  which  is  characterized  by  the  formation  of 
tubercular  thickenings  of  the  skin  and  tumefaction  of  the  nasal  mucous 
membrane,  followed  sometimes  by  ulceration  It  prevails  in  Italy,  Austria, 
and  to  a  slight  extent  in  some  parts  of  Germany.  Pathologists  generally 
regard  it  as  an  infectious  process,  although  this  has  not  been  proved. 

The  bacilli,  first  described  by  Von  Frisch,  appear  to  be  constantly  present 
in  the  newly  formed  tubercles.  They  are  commonly  found  in  certain  large 


FIG.  128.— Bacillus  of  rhinoscleroma  in  lymphatic  vessels  of  the  superficial  part  of  tumor. 
X  1,200.    CCornil  and  Babes  ) 

hyaline  cells  peculiar  to  the  disease,  and  may  also  be  observed  in  the  lym- 
phatic vessels  or  scattered  about  in  the  involved  tissues. 

Morphology. — Short  bacilli  with  rounded  ends,  usually  united  in  pairs, 
and  surrounded  by  a  gelatinous  capsule  resembling  that  of  Fried  lander's 
bacillus.  According  to  Eisenberg,  the  bacilli  are  two  to  three  times  as  long 
as  broad,  and  may  ^row  out  into  filaments. 

These  bacilli  stain  readily  with  the  aniline  colors  and  by  Gram's  method. 
The  capsule  may  be  demonstrated  by  the  methods  usually  employed  in  stain- 
ing Friedlander's  bacillus,  or  by  the  following  method  which  is  especially 
recommended  by  Alvarez:  The  excised  portions  of  tissue  involved  in  the  dis- 
ease are  placed  for  twenty-four  hours  in  a  one-per-cent  solution  of  osmic 
acid  and  then  in  absolute  alcohol.  When  properly  hardened  thin  sections 
are  made;  these  are  stained  in  a  hot  solution  of  aniline- water-methyl-violet 
for  a  few  minutes,  and  then  decolorized,  by  Gram's  method,  in  iodine  so- 
lution. 

Biological  Characters. — An  aerobic,  non-motile,  non-liquefying  bacillus 
(facultative  anaerobic  ?). 

In  gelatin  stick  cultures  the  growth  resembles  that  of  Friedlander's  ba- 
cillus— i.e.,  a  nail-like  growth,  consistingof  densely  crowded,  opaque  colonies 
along  the  line  of  puncture,  and  a  heaped-up,  white,  glistening  mass  upon  the 
surface,  hemispherical  in  form  and  viscous  in  consistence.  Upon  gelatin 
plates  yellowish-white,  spherical  colonies  are  developed  within  two  or  three 
days,  which  under  the  microscope  are  seen  to  be  granular.  Upon  potato  a 
cream-like  growth  occurs  along  the  line  of  inoculation,  which  is  white  or 
30 


426  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

yellowish-white  in  color,  and  in  which  gas  bubbles  may  be  developed.  De- 
velopment is  most  rapid  at  a  temperature  of  35°  to  38°,  but  also  occurs  at  the 
room  temperature. 

Pathogenesis. — The  etiological  relation  of  this  bacillus  to  the  disease  with 
which  it  is  associated  has  not  been  established.  It  is  pathogenic  for  mice 
and  for  guinea  pigs,  less  so  for  rabbits;  in  this  regard,  as  in  its  morphology 
and  growth  in  various  culture  media,  it  bears  a  close  resemblance  to  Fried- 
lander's  bacillus,  which  is  also  found  not  infrequently  in  the  nasal  secretions 
of  healthy  persons  and  in  those  suffering  from  chronic  nasal  catarrh  or  ozaena. 

The  principal  points  of  difference,  as  pointed  out  by  Baum^arten,  are  as 
follows:  The  bacillus  of  rhinoscleroma  is  usually  more  decidedly  rod  shaped 
than  Friedlander's  bacillus,  although  both  may  be  of  so  short  an  oval  as  to 
resemble  micrococci.  The  first-mentioned  bacillus  constantly  presents  the 
appearance  of  being  surrounded  by  a  transparent  capsule,  even  in  the  cul- 
tures in  artificial  media,  while  Friedlander's  bacillus  in  such  media  does  not 
usually  present  this  appearance,  unless  as  a  result  of  special  treatment. 
Finally,  the  bacillus  of  rhinoscleroma  may  retain  its  color,  in  part  at  least, 
when  treated  by  Gram's  method,  while  Friedlander's  bacillus  is  completely 
decolorized  when  placed  in  the  iodine  solution  employed  in  this  method. 

Notwithstanding  these  points  of  difference,  Baumgarten  is  not  entirely 
satisfied  that  this  bacillus  is  a  distinct  species,  and  several  bacteriologists 
have  maintained  that  it  is  identical  with  the  bacillus  of  Friedlander. 

59.    BACILLUS    OF    KOUBASOFF. 

Obtained  by  Koubasoff  (1889)  from  new  growths  in  the  stomach  of  a 
person  who  died  of  cancer  of  the  stomach. 

Morphology. — Bacilli  with  round  ends,  or  with  one  end  pointed,  two  or 
three  times  as  long  as  the  tubercle  bacillus  and  three  or  four  times  as  thick. 

Stains  readily  with  the  aniline  colors. 

Biological  Characters. — A.n  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Forms  spores  in  the  centre  of  the  rods.  Grows 
in  the  usual  culture  media  at  the  room  temperature,  more  rapidly  at  3<T  O. 
In  stick  cultures  in  glycerin-gelatin  the  growth  resembles  an  inverted  stetho- 
scopa;  at  the  surface  a  circular,  bluish  membrane  is  formed,  which  is  de- 
pressed in  the  form  of  a  funnel,  while  along  ths  line  of  puncture  a  slender, 
yellowish,  jagged  column  is  developed.  Upon  agrar,  at  36°  C.,  a  bluish- 
white  layer  is  quickly  developed.  Upon  potato  the  growth  resembles  that 
of  the  typhoid  bacillus  at  first;  later  a  granular  membrane  is  formed;  under 
a  low  power  the  granules  appear  to  be  formed  of  intertwined  masses  of  fila- 
ments. The  growth  upsn  blood  seru'n  is  similar  to  that  upon  agar. 

Pathogenesis. — Subcutaneous  injections  in  guinea-pigs  cause  their  death 
in  one  to  two  weeks,  in  rabbits  in  one  to  two  months,  in  cats  and  dogs  in 
two  months  or  more.  Death  occurs  in  a  shorter  time  in  animals  which  have 
been  fed  upon  cultures  than  as  a  result  of  subcutaneous  injections.  The 
animals  became  very  much  emaciated  and  have  paralysis  of  the  sphincter 
muscles.  At  the  autopsy  fiat  or  nodular  elevations,  which  are  often  ulce- 
rated, are  seen  here  and  there  upon  the  mucous  membrane  of  the  stomach 
and  intestine;  the  mesentery,  especially  of  the  small  intestine,  ishyperaemic; 
the  mesenteric  glands  are  swollen,  as  are  also  the  inguinal  glands.  In  the 
liver  and  sometimes  in  the  ovary,  uterus,  and  spleen  larger  or  smaller  nod- 
ules are  seen. 

60.    BACILLUS  OP  NOCARD. 

Obtained  by  Nocard  (1888)  from  pus  collected  from  the  superficial  ab- 
Q  rattle  Buffering  fro  n  a  ehrmic  infectious  disease  which  prevails 
especially  upon  the  island  of  Guadaloupe— known  as  4' farcin  du  boeuf" 
''•"'.    ••NVimnkraiiklu'it." 

Morphology.--*,  long  and  slender  bacillus,  about  as  thick  as  the  bacillus 
i    (Bacillus  murisepticus) ;  usually  seen  in  tangled  masses  which 
•  an  opaque  central  portion  surrounded  by  long  filaments,  which 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  427 

apparently  give  off  lateral  ramifications.  (This  description  of  the  morphol- 
ogy gives  rise  to  the  suspicion  that  the  microorganism  described  by  Nocard 
is  a  microscopic  fungus  rather  than  a  bacillus.)  According  to  Nocard,  the 
branching  is  more  apparent  than  real,  and  is  in  fact  a  false  cuchotomization, 
such  as  is  seen  in  the  genus  Cladothrix. 

Stains  best  by  Weigert's  method ;  is  decolorized  by  Gram's  method.  Does 
not  stain  readily  with  most  aniline  colors. 

Biological  Characters. — An  aerobic,  non-motile  bacillus,  which  does 
not  grow  in  nutrient  gelatin  at  the  room  temperature.  Grows  in  the  usual 
culture  media  at  a  temperature  of  30 3  to  40°  C.  Forms  small  oval  spores. 
Is  destroyed  in  ten  minutes  by  a  temperature  of  70°  C.  Upon  the  surface 
of  agar  it  forms  irregular,  opaque,  yellowish-white  colonies,  which  are 
thickest  at  the  margin,  have  a  dull,  dusty- looking,  mammillated  surface, 
and  after  a  time  become  confluent,  forming  a  thick,  wrinkled,  membranous 
layer.  Upon  potato  development  is  rapid  in  the  form  of  prominent,  dry, 
pale- yellow  plaques.  In  bouillon  whitish  flocculi  are  formed,  most  of  which 
fail  to  the  bottom,  while  some  float  upon  the  surface,  where  they  form  dry, 
dusty-looking,  rounded  pellicles  of  a  dirty-gray  color  with  a  greenish  reflection. 

Pathogenesis. — The  guinea-pig  is  the  most  susceptible  animal.  When 
injected  into  th^  peritoneal  cavity  of  one  of  these  animals  it  produces,  in 
from  nine  to  twenty  days,  lesions  which  closely  resemble  those  of  miliary 
tuberculosis.  At  the  autopsy  the  peritoneum  is  found  to  be  covered  with 
nodules,  in  the  centre  of  which  the  bacillus  is  found  in  tangled  masses ;  the 
liver,  spleen,  kidneys,  and  intestine  are  also  studded  with  pseudo-tubercles, 
but  these  are  only  found  in  the  peritoneal  coat  and  not  in  the  parenchyma 
of  the  various  organs,  or  in  the  organs  of  the  thoracic  cavity.  Intravenous 
injections  give  rise  to  lesions  similar  to  those  of  general  miliary  tuberculo- 
sis, the  organs  generally  containing  a  considerable  number  of  nodules,  in 
the  centre  of  which  tufts  of  bacilli  are  found.  In  cattle  and  sheep  similar 
lesions  result  from  intravenous  injections,  but  without  causing  the  death  of 
the  animal.  The  dog,  the  cat,  the  horse,  the  ass,  and  the  rabbit  are  immune. 
Subcutaneous  inoculations  in  guinea  pigs  produce  an  extensive  local  abscess, 
followed  by  a  chronic  induration  of  the  neighboring  lymphatic  glands. 


XII. 

BACILLI  WHICH  PRODUCE  SEPTICAEMIA  IN 
SUSCEPTIBLE  ANIMALS. 

WHEN,  as  a  result  of  accidental  (natural)  or  experimental  inocula 
tion,  a  microorganism  is  introduced  into  the  body  of  a  susceptible 
animal  which  is  able  to  multiply  in  its  blood,  producing  a  general  in- 
fection, we  speak  of  this  general  blood  infection  as  a  septicaemia. 
When  pathogenic  microorganisms  which  are  unable  to  multiply  in 
the  blood  establish  themselves  hi  some  particular  locality  in  the  ani- 
mal body  which  is  favorable  for  their  growth,  and  by  the  formation 
of  toxic  products,  which  are  absorbed,  give  rise  to  general  symptoms 
•of  poisoning,  we  designate  the  affection  toxaemia.  As  examples  of 
this  mode  of  pathogenic  action  we  may  mention  diphtheria  and 
tetanus.  As  a  rule,  the  various  forms  of  septicaemia  are  quickly 
fatal,  and,  as  the  microorganisms  to  which  they  are  due  multiply  in 
the  blood  of  the  infected  animal,  this  fluid  possesses  infectious  pro- 
perties, and,  when  inoculated  in  the  smallest  quantity  into  another 
susceptible  animal,  reproduces  the  same  morbid  phenomena.  A  typi- 
cal example  of  this  class  of  diseases  is  found  in  anthrax,  to  which 
disease  a  special  section  has  already  been  devoted  (VII.).  But  in 
this  and  other  forms  of  septicaemia  subcutaneous  inoculations  do  not, 
as  a  rule,  result  in  the  immediate  invasion  of  the  blood  by  the  para- 
sitic microorganism.  Often  a  local  inflammatory  process  of  consider- 
able extent  is  first  induced  ;  and  in  some  cases  general  infection  only 
occurs  a  short  time  before  the  death  of  the  animal,  depending,  per- 
haps, upon  a  previous  toxaemia  from  the  absorption  of  toxic  products 
developed  at  the  seat  of  local  infection.  The  pathogenic  action,  then, 
in  acute  forms  of  septicaemia  appears  to  result,  not  alone  from  the 
presence  and  multiplication  of  the  pathogenic  microorganism  in  the 
blood,  but  also  from  the  toxic  action  of  products  evolved  during  its 
growth. 

Some  of  the  pathogenic  bacilli  of  this  class  now  known  to  bac- 
teriologists have  been  discovered  by  studying  the  infectious  diseases 
induced  by  them  in  lower  animals  among  which  these  diseases  pre- 
vail naturally — i.e.,  independently  of  human  interference.  Many 


BACILLI    WHICH    PRODUCE   SEPTICAEMIA.  429 

more  are  known  to  us  from  experiments  made  in  pathological  labora- 
tories, in  testing  by  inoculations  into  animals  bacteria  obtained  from 
various  sources,  with  reference  to  their  pathogenic  power.  We  in- 
clude in  this  group  only  those  bacilli  which  induce  fatal  septicaemia 
in  susceptible  animals  when  injected  into  the  circulation  or  sub- 
cutaneously  in  a  comparatively  small  quantity — e.g.,  less  than  half 
a  cubic  centimetre  of  a  bouillon  culture. 

01.    BACILLUS    SEPTICAEMIA   H^EMORRHAGIC^E. 

Synonyms. — Bacillus  of  fowl  cholera ;  Microbe  du  cholera  des 
poules  (Pasteur) ;  Bacillus  cholerae  gallinarum  (Fliigge) ;  Bacillus  der 
Hiihnercholera ;  Bacillus  of  rabbit  septicaemia  ;  Bacillus  cuniculi- 
cida  (Fliigge)  ;  Bacillus  der  Kaninchenseptikamie  (Koch)  ;  Bacillus 
der  Binderseuche  (Kitt)  ;  Bacillus  der  Schweineseuche  (Loffler  and 
Schutz)  ;  Bacillus  der  Wildseuche  (Hueppe)  ;  Bacillus  der  Biiffel- 
seuche  (Oreste-Armanni)  ;  (Bacterium  of  Davaine's  septicaemia  ?) 

It  is  now  generally  admitted  by  bacteriologists  that  Koch's  ba- 
cillus of  rabbit  septicaemia  (1881)  is  identical  with  the  bacillus 
("micrococcus")  of  fowl  cholera  previously  described  by  Pasteur 
(1880).  The  similar  bacilli  found  in  the  blood  of  animals  dead  from 
the  infectious  diseases  known  in  Germany  as  Wildseuche  (Hueppe), 
Rinderseuche  (Kitt),  Schweineseuche  (Schutz),  and  Buffelseuche 
(Oreste-Armanni)  appear  also  to  be  identical  with  the  bacillus  of 
rabbit  septicaemia  and  fowl  cholera.  This  view  is  sustained  by 
Hueppe  and  by  Baumgarten,  and  by  the  comparative  researches  of 
Caneva  (1891)  and  of  Bunzl-Federn  (1891). 

This  is  evidently  a  widely  distributed  pathogenic  bacillus  ;  it  was 
obtained  by  Koch  from  rabbits  inoculated  with  pu- 
trefying flesh  infusion,  by  Gaffky  from  impure  river 
water,  and  by  Pasteur  from  the  blood  of  fowls  suffer- 
ing from  the  infectious  disease  known  in  France  as 
cholera  des  poules.  It  is  not  infrequently  found  in 
putrefying  blood,  and  its  presence  in  the  salivary 
secretions  of  man  has  occasionally  been  demonstrated  FlQ  l>29  Bacinus 

(Baumgarten).  Septica!m.i89^b8tf^." 

With  reference  to  the  American  swine  plague 
described  by  Salmon  and  Smith,  we  are  informed  by 
Smith,  in  his  most  recent  publication  upon  the  subject 
(Zeitschrift  fur  Hygiene,  Band  x.,  page  493),  that  cultures  of  the 
German  Schweineseuche  bacillus,  received  from  the  Berlin  Hygienic 
Institute,  compared  with  his  cultures  from  infected  swine  in  this 
country,  agreed  in  all  particulars,  except  that  the  former  were  de- 
cidedly more  pathogenic  for  swine  and  for  rabbits. 

It  appears  extremely  probable  that  the  form  of  septicaemia  studied 


430  BACILLI   WHICH   PRODUCE  SEPTIOSSMIA 

by  Davaine  (1872),  which  he  induced  in  the  first  instance  by  inject- 
ing putrid  ox  blood  into  rabbits,  was  due  to  the  same  pathogenic  ba- 
cillus. The  writer  obtained  this  bacillus  (1887)  in  Cuba  from  the 
blood  of  rabbits  inoculated  with  liver  tissue  taken  from  a  yellow- 
fever  cadaver  and  kept  for  forty-eight  hours  in  an  antiseptic  wrap- 
ping. The  name  which  we  have  adopted  is  that  proposed  by  Hueppe 
for  the  form  of  septicaemia  to  which  it  gives  rise — "Septikamia 
hamorrhagica. " 

Morphology. — Short  bacilli  with  rounded  ends,  from  0.6  to  0.7 
p  in  diameter  and  about  1.4  ft  long;  sometimes  united  in  pairs,  or 
in  chains  of  three  or  four  elements.  In  stained  preparations  the  ex- 
tremities are  usually  stained,  while  the  central  portion  of  the  rod 
remains  unstained.  This  "  end  staining"  causes  the  rods  to  present 
the  appearance  of  diplococci  when  examined  with  a  comparatively 
low  power,  and  some  of  the  earlier  observers  described  the  microor- 
ganism under  consideration  as  a  micrococcus.  It  is  quickly  stained 
by  the  aniline  colors  usually  employed,  but  loses  its  color  when 
treated  by  Gram's  method. 

Biological  Characters. — A  non-motile,  aerobic,  non-liquefy- 
ing bacillus.  Does  not  form  spores.  Grows  in  various  culture  media 
at  the  room  temperature,  but  more  rapidly  at  35°  to  37°  C. — the 
lowest  temperature  at  which  development  occurs  is  about  13°  C. 
Although  this  is  an  aerobic  bacillus  and  a  certain  amount  of  oxygen 
is  necessary  for  its  development,  it  appears  to  grow  better  when  the 
amount  is  somewhat  restricted  than  it  does  on  the  surface  of  nutrient 
media. 

Upon  gelatin  plates,  at  the  end  of  two  or  three  days,  small, 
white  colonies  are  developed  upon  or  near  the  surface ;  these  are 
finely  granular  and  spherical,  with  a  more  or  less  irregular  outline, 
and  by  transmitted  light  have  a  yellowish  color  ;  later  the  central 
portion  of  the  colonies  is  of  a  yellowish-brown  color  and  is  sur- 
rounded by  a  transparent  peripheral  zone.  The  superficial  colonies 
are  commonly  smaller  than  those  which  develop  a  little  below  the 
surface  of  the  gelatin.  In  stick  cultures  in  nutrient  gelatin  the 
growth  upon  the  surface  consists  of  a  thin,  whitish  layer  in  the 
vicinity  of  the  point  of  puncture,  having  an  irregular,  jagged  out- 
line— sometimes  there  is  no  development  upon  the  surface ;  along 
the  line  of  puncture  the  growth  consists  of  rather  transparent,  dis- 
crete or  confluent  colonies.  In  streak  cultures  upon  nutrient  agar, 
or  gelatin,  or  blood  serum  the  growth  is  limited  to  the  immediate 
vicinity  of  the  line  of  inoculation,  and  consists  of  finely  granular, 
semi-transparent  colonies,  which  form  a  thin,  grayish-white  layer 
with  irregular,  somewhat  thickened  margins.  Upon  potato  no  de- 
velopment occurs,  as  a  rule,  at  the  room  temperature,  but  in  the  in- 


IN  SUSCEPTIBLE  ANIMALS. 


43 1 


cubating  oven  a  rather  thin,  transparent,  grayish-white  or  yellowish, 
waxy  layer  is  developed  in  the  course  of  a  few  days.     According  to 
Bunzl-Federn,  the  bacillus  of  fowl  cholera  and  that 
of  rabbit  septicaemia  grow  upon  potato,  while  the 
bacillus  of  Wildseuche,  Schweineseuche,  and  Biif- 
felseuche  do  not.     According  to 
Caneva,  none  of  the  bacilli  of  this 
group  grow  upon  potato.      The 
same  author  states  that  the  growth 
in  milk  is  scanty  and  does  not 
produce  coagulation,  while  Bunzl- 
Federn  finds  that  the  bacillus  of 
fowl  cholera  and  of  rabbit  septi- 
caemia produce  coagulation  and 
the  others  do  not.     These  differ- 
ences are  not,  however,  consid- 
ered by  the  author  last  named  as 
sufficient  to  establish  the  specific 
difference  of  the  bacilli  from  these 
diff erent  sources.    He  looks  upon 
them  rather  as  varieties  of  the 
same  species.     Bunzl-Federn  has 
also  ascertained  that  when  cul- 
tivated in  a  peptone  solution  all 
of  the  bacilli  of  this  group,  with 
the  exception  of    that  obtained 
from  the  so-called  Buffelseuche, 
give  the  reaction  for  phenol  and 
for  indol— the  bacillus  of  Buffel- 
seuche gives  the  indol  reaction  only.    Development  in  bouillon  is  rapid 
and  causes  a  uniform  turbidity  of  the  fluid.     Cultures  of  this  bacillus 
may  retain  their  vitality  for  three  months  or 
more  when  kept  in  a  moist  condition ;  but 
the  bacillus  usually  fails  to  grow  after  having 
been  kept  for  a  few  days  in  a  desiccated  con- 
dition ;  according  to  Hueppe,  it  may  resist 
desiccation  for  fourteen  days.     The  thermal 
death-point,  as  determined  by  Salmon  for 
the  bacillus  of  fowl  cholera,  is  56°  C. ,  the  time 
of  exposure  being  ten  minutes  (55°  C.  with 
fifteen  minutes'  exposure — Baumgarten).     It 
is  not  readily  destroyed  by  putrefaction  (Kitt). 
A  solution  of  mercuric  chloride  of   1  :5,000 
destroys  it  in  one  minute,  and  a  three-per-cent  solution  of  carbolic 


PIG.  130. —Bacillus 
septicaemias  haemor- 
rhagicae;  stick  culture 
in  nutrient  gelatin, 
end  of  four  days  at  16°- 
18°  C.  (Baumgarten  ) 


Fia.  131.— Bacillus 
of  Schweineseuche  ; 
old  stick  culture 
in  nutrient  gela- 
tin. (Schutz.) 


FIG.  132.  —  Bacillus  of  swine 
plague;  colonies  on  gelatin 
plate,  end  of  seven  days. 
X  00.  (Smith.) 


432  BACILLI  WHICH   PRODUCE   SEPTICAEMIA 

acid  in  six  hours  (Hueppe).  Pasteur  (1880)  has  shown  that  when 
cultures  of  this  bacillus  (microbe  of  fowl  cholera)  in  bouillon  are 
kept  for  some  time  they  gradually  lose  their  pathogenic  virulence, 
and  he  has  ascribed  this  " attenuation  of  virulence"  to  the  action  of 
atmospheric  oxygen.  He  also  ascertained  that  the  particular  degree 
of  \irulence  manifested  by  the  mother  culture  after  a  certain  interval 
could  be  maintained  in  successive  cultures  made  at  short  intervals. 
He  was  thus  able  to  cultivate  different  pathogenic  varieties,  and  to 
use  these  in  making  protective  inoculations,  by  which  susceptible  ani- 
mals were  preserved  from  the  effects  of  virulent  cultures  injected 
subsequently. 

Attenuated  cultures  recover  their  virulence  when  inoculated  into 
very  susceptible  animals.  Thus  a  culture  which  would  produce  a 
non-fatal  and  protective  attack  in  a  chicken  may,  according  to  Pas- 
teur, kill  a  small  bird,  like  a  sparrow;  and  by  successive  inoculations 
from  one  sparrow  to  another  the  original  degree  of  virulence  may  be 
restored,  so  that  a  minute  quantity  of  a  pure  culture  would  be  fata' 
to  a  chicken. 

Pathogenesis. — Pathogenic  for  chickens,  pigeons,  pheasants, 
sparrows,  and  other  small  birds,  for  rabbits  and  mice,  also  for  swine 
(Schweineseuche),  for  cattle  (Rinderseuche),  and  for  deer  (Wild- 
seuche).  Subcutaneous  injection  of  a  minute  quantity  of  a  virulent 
culture  usually  kills  chickens  within  forty-eight  hours.  Some  time 
before  death  the  fowl  falls  into  a  somnolent  condition,  and,  with 
drooping  wings  and  ruffled  feathers,  remains  standing  in  one  place 
until  it  dies.  Infection  may  also  occur  from  the  ingestion  of  food 
moistened  with  a  culture  of  the  bacillus  or  soiled  with  the  discharges 
from  the  bowels  of  other  infected  fowls.  At  the  autopsy  the  mucous 
metnbrane  of  the  small  intestine  is  found  to  be  inflamed  and  studded 
with  small  hsemorrhagic  foci,  as  are  also  the  serous  membranes ;  the 
spleen  is  notably  enlarged.  The  bacilli  are  found  in  great  numbers 
in  the  blood,  in  the  various  organs,  and  in  the  contents  of  the  in- 
testine. In  rabbits  death  commonly  occurs  in  from  sixteen  to  twenty 
hours,  and  is  often  preceded  by  convulsions.  The  temperature  is 
elevated  at  first,  but  shortly  before  death  it  is  reduced  below  the 
normal.  The  post-mortem  appearances  are  :  swelling  of  the  spleen 
and  lymphatic  glands  ;  ecchymoses  or  diffuse  haBmorrhagic  infiltra- 
tions of  the  mucous  membranes  of  the  digestive  and  respiratory  pas- 
sages, and  in  the  muscles  ;  and  at  the  point  of  inoculation  a  slight 
amount  of  inflammatory  oedema.  The  bacilli  are  found  in  consider- 
able numbers  in  the  blood  within  the  vessels,  or  in  that  which  has 
escaped  into  the  tissues  by  the  rupture  of  small  veins.  They  are  not, 
however,  so  numerous  as  in  some  other  forms  of  septicaemia — e.g., 
Anthrax,  mouse  septicaemia — when  an  examination  is  made  imme- 


IN   SUSCEPTIBLE   ANIMALS.  433 

diately  after  death  ;  later  the  number  may  be  greatly  increased  as  a 
result  of  post-mortem  multiplication  within  the  vessels.  The  rabbit 
is  so  extremely  susceptible  to  infection  by  this  bacillus  that  inocula- 
tion in  the  cornea  by  a  slight  superficial  wound  usually  gives  rise  to 
general  infection  and  death.  This  animal  may  also  be  infected  by 
the  ingestion  of  food  contaminated  with  a  culture  of  the  bacillus.  It 
is  by  this  means  that  Pasteur  proposed  to  destroy  the  rabbits  in  Aus- 
tralia, which  have  multiplied  in  that  country  to  such  an  extent  as  to 
constitute  a  veritable  pest.  Both  in  fowls  and  in  rabbits  the  disease 
may  under  certain  circumstances  run  a  more  protracted  course — e.g., 
when  they  are  inoculated  with  a  small  quantity  of  an  attenuated  cul- 
ture. In  less  susceptible  animals — guinea-pigs,  sheep,  dogs,  horses 


FIG.  138.— Bacillus  of  Sahweineseuche,  in  blood  of  rabbit.    (Schutz.) 

— a  local  abscess,  without  general  infection,  may  result  from  the  sub- 
cutaneous injection  of  the  bacillus  ;  but  these  animals  are  not  entirely 
immune.  In  the  infectious  maladies  of  swine,  cattle,  deer,  and  other 
large  animals  to  which  reference  has  been  made,  and  which  are  be- 
lieved to  be  due  to  the  same  bacillus,  the  symptoms  and  pathological 
appearances  do  not  entirely  correspond  with  those  in  the  rabbit  or 
the  fowl;  but  the  bacillus  as  obtained  from  the  blood  of  such  animals 
corresponds  in  its  morphological  and  biological  characters  with  Pas- 
teur's microbe  of  fowl  cholera  and  Koch's  bacillus  of  rabbit  septi- 
caemia, and  pure  cultures  from  the  various  sources  mentioned  are 
equally  fatal  to  rabbits  and  to  fowls.  In  the  larger  animals  pul- 
monary and  intestinal  lesions  are  developed,  and  in  swine  a  diffused 
red  color  of  the  skin,  similar  to  that  observed  in  the  disease  known 
in  Germany  as  Schweinerothlauf  (Fr.  rouget),  is  sometimes  seen. 


434  BACILLI   WHICH    PRODUCE   SEPTICAEMIA 

According  to  Baumgarten,  bacilli  from  Wildseuche  or  from  Kinder- 
seuche  inoculated  into  swine  give  rise  to  fatal  Schweineseuche,  and 
bacilli  from  any  of  these  forms  of  disease,  when  inoculated  into 
pigeons,  produce  characteristic  fowl  cholera  ;  but  the  bacillus  as  ob- 
tained from  Schweineseuche  or  Wildseuche  is  not  fatal  to  chickens, 
and  the  bacillus  from  Schweineseuche  is  fatal  to  guinea-pigs,  which 
have  but  slight  susceptibility  to  the  bacillus  of  rabbit  septicaemia. 
Notwithstanding  these  differences  he  agrees  with  Hueppe  in  the  view 
that  the  bacilli  from  the  various  sources  mentioned  are  specifically 
identical ;  although  evidently,  if  this  view  is  adopted,  we  must 
admit  that  varieties  exist  which  differ  somewhat  in  their  pathogenic 
power. 

The  researches  of  Smith  and  of  Moore  show  that  "  an  attenuated 
variety  of  bacteria,  belonging  to  the  group  of  swine-plague  bacteria 
and  not  distinguishable  from  them,  inhabit  the  mouth  and  upper  air 
passages  of  such  domesticated  animals  as  cattle,  dogs,  and  cats" 
(Smith). 

62.    BACILLUS   OF   CHOLERA   IN   DUCKS. 

Obtained  by  Cornil  and  Toupet  (1888)  from  the  blood  of  ducks,  in  the 
Jardin  d'Acclimation  at  Paris,  which  had  died  of  an  epidemic  disease  charac- 
terized by  diarrhoea,  feebleness,  and  muscular  tremors,  and  which  resulted 
fatally  in  two  or  three  days. 

Morphology. — Does  not  differ  in  its  morphology  from  the  bacillus  of 
fowl  cholera  (Bacillus  septicaemias  haemorrhagicse) ;  short  rods  with  rounded 
ends,  from  1  to  1.5  ft  in  length  and  0.5  //  broad. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method ;  the  ends 
stain  more  deeply  than  the  central  portion. 

Biological  Characters.— An  aerobic,  non  liquefying,  non- motile  bacillus. 
Does  not  form  spores.  Grows  in  the  usual  culture  media  at  the  room  tem- 
peratu  re.  In  its  gro  wth  in  various  media,  as  well  as  in  its  morphology,  Cornil 
and  Toupet  found  this  bacillus  to  correspond  with  the  bacillus  of  fowl 
cholera.  In  gelatin  stick  cultures  the  growth  upon  the  surface  consists  of  a 
thin,  gravish  layer,  and  along  the  line  of  puncture  as  small,  semi  transpa- 
rent, slightly  yellowish,  spherical  colonies.  Upon  agar,  in  the  incubating 
oven,  at  the  end  of  twelve  hours  small,  lentil  shaped,  waxy  colonies  are 
formed,  which  later  may  have  a  diameter  of  three  to  four  millimetres. 
Upon  potato  circular,  yellowish  colonies  are  formed,  which  become  con- 
fluent and  form  a  somewhat  depressed,  pale-yellow  layer. 

Pathogenesis. — According  to  Cornil  and  Toupet,  this  bacillus  is  patho- 
genic for  ducks,  but  not  for  chickens  or  pigeons,  and  only  kills  rabbits  when 
injected  in  considerable  quantity.  Ducks  die  in  from  one  to  three  days 
from  subcutaneous  injections,  or  by  the  ingestion  of  food  to  which  the  bacil- 
lus has  been  added. 

63.  BACILLUS  OP  HOG  CHOLERA  (Salmon  and  Smith). 

Synonyms. — Bacillus  of  swine  plague  (Billings) ;  Bacillus  of  swine- 
pest  (Selander). 

According  to  Smith,  this  bacillus  was  first  described  by  Klein 
(1884)  ;  it  was  first  obtained  in  pure  cultures  and  its  principal  char- 
acters determined  by  Salmon  and  Smith  (1885),  and  has  since  been 


IN  SUSCEPTIBLE   ANIMALS.  435 

studied  in  cultures  and  by  experimental  inoculations  by  Selander, 
Billings,  Frosch,  Welch,  Caneva,  Bunzl-Federn,  and  others. 

The  bacillus  is  found  in  the  blood  and  various  organs  of  hogs 
which  have  succumbed  to  the  infectious  disease  known  in  this  country 
as  hog  cholera ;  and  also  in  the  contents  of  the  intestine,  from  which 
it  may  be  obtained  by  inoculations  into  rabbits,  but  is  not  easily  iso- 
lated by  the  plate  method  owing  to  the  large  number  of  other  bac- 
teria present  (Smith). 

Morphology. — Short  bacilli  with  rounded  ends,  1.2  to  1.5  yu  in 
length  and  0.6  to  0.7  /*  in  breadth  ;  usually  united  in  pairs. 

This  bacillus  is  easily  stained  by  the  aniline  colors  usually  em- 
ployed, but  does  not  retain  its  color  when  treated  by  Gram's  method. 
When  the  staining  agent  is  allowed  to  act  for  a  very  short  time  the 


FIG.  131.— Bacillus  of  hog  cholera;  stained  by  Loffler's  method  to  show  flagella.  x  1,000.  From 
a  photomicrograph  made  at  the  Army  Medical  Museum.  (Gray.) 

ends  of  the  rods  may  be  stained  while  the  central  portion  remains 
unstained. 

Biological  Characters. — An  aerobic  (facultative  anaerobic),  non- 
liquefying,  actively  motile  bacillus.  In  many  of  its  characters  this 
bacillus  closely  resembles  the  one  last  described  (Bacillus  septicaemias 
hsemorrhagicse),  but  it  is  distinguished  from  it  by  its  active  move- 
ments, which,  according  to  Smith,  may  be  still  observed  in  cultures 
which  have  been  kept  for  weeks  or  months.  Does  not  form  spores. 
Grows  readily  in  various  culture  media  at  the  room  temperature — 
more  rapidly  in  the  incubating  oven.  Upon  gelatin  plates  colonies 
are  developed  in  from  twenty-four  to  forty-eight  hours.  The  deep  colo- 
nies are  spherical  and  homogeneous,  and  have  a  brownish  color  by 
transmitted  light;  they  seldom  exceed  one-half  millimetre  in  diameter. 


436  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

The  superficial  colonies  may  attain  a  diameter  of  two  millimetres  : 
they  present  no  distinctive  characters.  Upon  agar  plates  the  colonies 
may  have  a  diameter  of  four  millimetres  ;  they  have  a  grayish,  trans- 
parent appearance  and  a  shining  surface.  In  gelatin  stick  cultures 
small,  yellowish-white  colonies  are  developed  along  the  line  of  in  - 
oculation,  which  may  become  confluent ;  upon  the  surface  a  thin. 
pearly  layer  is  developed  about  the  point  of  inoculation,  which  may 
have  a  diameter  of  six  millimetres  or  more.  Upon  potato  a  straw- 
yellow  layer  is  developed,  which  later  acquires  a  darker  color.  In 
slightly  alkaline  bouillon  a  slight  cloudiness  may  be  observed  at  the 
end  of  twenty -four  hours,  and  at  the  end  of  one  or  two  weeks,  if 
not  disturbed,  a  deposit  is  seen  at  the  bottom  of  the  tube  and  a  thin, 
broken  film  may  form  upon  the  surface.  The  development  of  this 
bacillus  in  milk  produces  a  direct  solution  of  the  casein  without  pre- 
vious coagulation  ;  when  a  solution  of  litmus  has  been  added  to  milk 
it  retains  its  blue  color  in  presence  of  this  bacillus,  while  the  bacillus 
previously  described  causes  it  to  change  to  red.  Neither  phenol 
nor  indol  is  produced  in  solutions  containing  peptone  (Bunzl-Federn) 
— another  distinguishing  character  from  the  Bacillus  septicaemias 
hsemorrhagicae.  This  bacillus  may  be  cultivated  in  slightly  acid 
media,  which  after  a  time  acquire  an  alkaline  reaction. 

In  Smith's  experiments  this  bacillus  was  found  to  resist  desicca- 
tion from  nine  days  to  several  months,  according  to  the  thickness  of 
the  layer  dried  upon  the  cover  glass  ;  bacilli  from  an  agar  culture  in 
some  experiments  failed  to  grow  after  seventeen  days,  and  in  others 
still  gave  cultures  after  four  months.  Bouillon  cultures  are  steril- 
ized in  four  minutes  by  a  temperature  of  70°  C.,  in  fifteen  minutes 
by  58°  C.,  and  in  one  hour  by  54°  C.  (Smith).  Novy  has  isolated 
from  cultures  of  the  hog-cholera  bacillus  a  toxic  basic  substance 
which  he  calls  susotoxin.  This  was  obtained  by  Brieger's  method  ; 
it  is  a  yellowish-brown,  syrup-like  liquid,  which,  when  injected  into 
rats  in  doses  of  0.125  to  0.25  cubic  centimetre,  causes  their  death  in 
less  than  thirty-six  hours.  He  also  obtained  by  precipitation  with 
absolute  alcohol,  from  cultures  concentrated  in  a  vacuum  at  36°  C., 
a  toxalbumin  which  when  dried  was  in  the  form  of  a  white  powder 
easily  soluble  in  water.  Rats  died  in  three  or  four  hours  after  re- 
ceiving subcutaneously  a  dose  of  0.1  to  0.5  gramme. 

Pathogenesis. — Pathogenic  for  swine,  rabbits,  guinea-pigs,  mice, 
and  pigeons. 

In  certain  parts  of  the  United  States  the  disease  known  as  "  hog 
cholera "  f requently  prevails  among  swine  as  a  fatal  epidemic.  It 
may  occur  as  an  acute  and  quickly  fatal  septicaemia,  or  in  a  more 
chronic  form  lasting  from  two  to  four  weeks  or  even  longer.  In 
the  acute  form  death  may  occur  within  twenty-four  hours,  and  haem- 


IN   SUSCEPTIBLE   ANIMALS.  437 

orrhagic  extravasations  are  found  upon  the  mucous  and  serous 
membranes  and  in  the  parenchyma  of  the  lungs,  kidneys,  and  lym- 
phatic glands.  The  spleen  is  greatly  enlarged,  soft,  and  dark  in 
color.  In  the  chronic  form  of  the  disease  the  most  notable  changes 
are  found  in  the  alimentary  canal.  These  are  most  constant  and 
characteristic  in  the  caecum  and  colon,  which  may  be  studded  with 
spherical,  hard,  necrotic  masses  or  extensive  diphtheritic  patches. 
According  to  Smith,  the  hsemorrhagic  and  necrotic  form  of  the  dis- 
ease may  exist  at  the  same  time  in  different  animals  of  the  same 
herd.  The  bacilli  are  found  in  all  of  the  organs,  and  especially  in 
the  spleen,  where  they  are  associated  in  irregular  colonies  similar 
to  those  of  the  typhoid  bacillus.  Smith  has  demonstrated  their  pre- 
sence in  urine  taken  from  the  bladder  immediately  after  the  death 
of  the  animal,  and  states  that  the  kidneys  are  almost  always  in- 
volved, as  shown  by  the  presence  of  albumin  and  tube  casts  in  the 
urine. 

An  extremely  minute  quantity  of  a  bouillon  culture  injected  be- 
neath the  skin  of  a  rabbit  causes  its  death  in  from  seven  to  twelve 
days  ;  a  larger  quantity  may  produce  a  fatal  result  in  five  days  ;  in- 
travenous injections  of  very  small  amounts  may  be  fatal  within 
forty-eight  hours.  After  a  subcutaneous  injection  the  animal  re- 
mains in  apparent  good  health  for  three  or  four  days,  after  which  it 
loses  its  appetite  and  is  indisposed  to  move  ;  several  days  before 
death  the  temperature  is  suddenly  elevated  from  2°  to  3°  C.,  and  it 
remains  high  until  the  fatal  termination.  At  the  autopsy  the  spleen 
is  found  to  be  enlarged  and  of  a  dark-red  color  ;  the  liver  is  studded 
with  small,  yellowish- white,  necrotic  foci;  the  kidneys  have  under- 
gone parenchymatous  changes  ;  the  heart  is  fatty  ;  and  the  intestinal 
mucous  membrane  is  more  or  less  marked  with  hsemorrhagic  extra- 
vasations. The  bacilli  are  found  in  all  of  the  organs.  In  house 
mice  the  results  of  experimental  inoculations  are  similar  to  those  in 
rabbits.  Guinea-pigs  succumb  when  inoculated  subcutaneously  with 
one-tenth  cubic  centimetre ;  pigeons  require  a  still  larger  dose — 
about  three-quarters  of  a  cubic  centimetre.  Swine  are  killed  by  the 
intravenous  injection  of  one  to  two  cubic  centimetres  of  a  recent 
bouillon  culture,  but,  as  a  rule,  do  not  succumb  to  subcutaneous 
injections.  Cultures  recently  obtained  from  diseased  animals  are 
more  virulent  than  those  which  have  been  propagated  for  a  consider- 
able time  in  artificial  media. 

Smith  has  described  a  variety  of  the  hog-cholera  bacillus  obtained  during- 
an  epidemic  in  which  the  disease  was  of  longer  duration — about  four  weeks 
—than  is  usual,  and  in  which  there  was  commonly  found  at  the  autopsy  a 
diphtheritic  inflammation  of  the  mucous  membrane  of  the  stomach.  This 
bacillus  differed  from  the  typical  form  by  being  somewhat  larger  and  in 
forming  considerably  larger  colonies  in  gelatin  plates — two  or  three  times 


438  BACILLI   WHICH   PRODUCE   SEPTIOEMIA 

as  large.  It  also  produced  a  greater  opacity  in  peptonized  bouillon,  and  in 
general  showed  a  more  vigorous  growth  in  various  nutrient  media.  It  dif- 
fered also  in  its  pathogenic  power,  as  tested  upon  rabbits,  causing-  death  at  a 
later  date  or  not  at  all ;  and  in  fatal  cases  the  swelling  of  the  spleen  and 
necrotic  foci  in  the  liver,  produced  by  the  first-described  species,  were  absent. 

Bang  (1892)  has  obtained  a  bacillus  from  infected  swine  in  Denmark 
which  corresponds  with  the  American  hog-cholera  bacillus.  In  chronic 
forms  of  the  disease  pneumonia  and  an  extensive  diphtheritic  process  in  the 
intestines  occurred  as  a  complication.  This  was  found  to  be  due  to  another 
bacillus,  called  by  Bang  "  vacuole-bacillus."  This  produced  a  fatal  pleuro- 
pneumonia  when  injected  into  the  lungs  in  pigs.  According  to  Bang,  his 
*4  vacuole-bacillus  "  is  without  doubt  identical  with  the  swine-plague  bacillus 
of  Salmon  and  Smith,  and  the  disease  of  swine  studied  by  him  was  a  mixed 
infection.  The  necrotic  changes  in  the  intestine,  found  in  cases  running  a 
chronic  course,  are  believed  by  Bang  to  be  due  to  still  another  bacillus—his 
"necrosis-bacillus."  Affanas'sieff  (1892)  confirms  the  results  previously  ob- 
tained by  several  independent  observers  as  to  the  identity  of  the  swine-plague 
bacillus  of  Salmon  and  Smith  with  the  Loffler-Schiitz  bacillus.  The  only 
difference  observed  was  a  difference  in  pathogenic  virulence — the  bacillus 
from  America  corresponding  with  a  somewhat  attenuated  variety  of  that 
from  Germany. 

Welch  (1894),  as  a  result  of  his  extended  researches,  arrives  at  the  follow- 
ing conclusion : 

"Our  own  conclusion  as  to  the  bacteria  of  Schweineseuche  and  of  swine 
plague  is  that  no  difference  exists  between  them  as  regards  morphology, 
culture  behavior,  and  pathogenic  effects  on  rabbits,  mice,  and  other  labora- 
tory animals.  Cultures  of  each  occur  which  are  also  indistinguishable  by 
inoculation  of  pigs.  The  only  difference  by  laboratory  experiment  which 
has  thus  far  been  brought  out  is  that  there  occur  Schweineseuche  bacilli  of 
higher  degree  of  virulence  as  tested  on  pigs  than  any  swine-plague  bac- 
teria which  have  hitherto  been  isolated  from  pigs  in  this  country.  Another 
point  to  be  considered  in  this  connection  is  that  Schweineseuche  occurs  as 
an  independent  disease  in  Germany  without  association  with  hog  cholera, 
whereas  swine  plague  has  not  been  shown  to  prevail  with  the  same  inde- 
pendence as  an  epizootic  in  this  country." 

Silberschmidt  (1895)  arrives  at  a  different  conclusion  from  that  reached 
by  Smith,  Welch,  Bang,  and  others,  He  believes  that  the  diseases  of  swine 
known  as  hog  cholera,  swine  plague,  and  infectious  pneumo-enteritis  are  all 
due  to  one  and  the  same  bacillus,  which,  however,  varies  considerably  both 
in  its  morphological  characters  and  its  pathogenic  power.  In  view  of  the 
results  previously  reached  by  equally  competent  bacteriologists,  and  especially 
by  Smith  and  by  Welch  in  this  country,  we  are  not  disposed  to  accept  the 
view  maintained  by  Silberschmidt. 

Smith  has  described  several  varieties  of  the  hog-cholera  bacillus,  and  in 
his  account  of  the  "hog-cholera  group  of  bacteria "  shows  that  the  Bacillus 
enteriditis  of  Gartner  and  the  Bacillus  typhi  murium  of  Loffler  belong  to 
this  group.  The  characters  of  the  different  varieties  (or  species?)  belonging 
to  the  group  are  given  by  Smith  in  detail  (United  States  Department  of  Agri- 
culture, Bureau  of  Animal  Industry,  Bulletin  No.  6,  1894),  and  the  follow- 
ing general  statement  is  made: 

"  If  we  attempt  to  sum  up  those  characters  which  are  to  circumscribe  the 
hog-cholera  group  of  bacteria  we  are  at  once  confronted  by  the  scarcity  of 
common  character's.  Pathogonesis,  though  of  great  importance  from  the 
standpoint  of  pathology,  is  probably  the  last  character  acquired  and 
evident  ly  the  most  variable  and  most  readily  lost.  If  we  base  the  unity 
of  this  group  on  morphological  and  biological  characters,  we  are  like- 
wise met  by  variations  in  size,  absence  of  motility,  variations  in  the  ap- 
pearancr  <>f  th«-  colonies.  Then*  are,  however,  certain  underlying  char- 


IN   SUSCEPTIBLE  ANIMALS.  439 

acters,  as  expressed  by  the  behavior  of  these  bacteria  in  bouillon  con- 
taining- dextrose,  saccharose,  and  lactose,  which  I  think  will  serve  as  a  very 
important  group  character,  differentiating  such  groups  sharply  from  the 
colon  group.  I  would  therefore  suggest  that  for  the  present  all  bacteria 
whose  size  approximates  that  of  this  group,  which  do  not  liquefy  gelatin,  and 
whose  fermentative  properties  are  the  same  as  those  described  for  this  group, 
should  be  ranged  under  it.  Future  investigations  into  the  biochemical  char- 
acters* of  these  varieties  or  sub-species  may  reveal  other  differential  charac- 
ters, but  the  time  has  not  yet  come  when  such  laborious  work  will  be  under- 
taken o»  a  sufficiently  extensive  scale  to  be  of  any  service  in  differentiating 
varieties  and  sub-species." 

Selander  in  1890,  and  Metschnikoff  in  1892,  have  reported  a  rapid  increase 
in  virulence  of  the  bacillus  of  hog  cholera  by  successive  inoculations  in 
rabbits*  or  pigeons.  Moore  (1894)  has  shown  that  this  is  a  mistake,  and  that 
the  bacteriologists  named  probably  did  not  experiment  with  cultures  of  the 
hog-cholera  bacillus,  as  they  supposed,  but  that  their  experiments  were 
made  with  the  bacillus  of  swine  plague — Bacillus  septicaemias  hemprrhagi- 
cae — which  when  passed  through  a  series  of  rabbits  attains  a  notable  increase 
in  pathogenic  virulence. 

In  a  recent  article,  Klein,  of  London  (1895)  says:  "  The  bacillus  of 
English  swine  plague,  which  I  described  in  1884,  in  Virchow's  Archiv,  as 
shown  by  Smith  and  Welch,  is  identical  with  the  bacillus  of  American  hog 
cholera." 


64.    BACILLUS   OF   BELFANTI   AND   PASCAROLA. 

Synonym.  — Impf  tetanusbacillus. 

Obtained  by  Belfanti  and  Pascarola  (1888)  from  the  pus  of  wounds  in  an 
individual  who  succumbed  to  tetanus. 

Morphology. — Bacilli  with  rounded  ends,  sometimes  so  short  as  to  resemble 
micrococci ;  resemble  the  Bacillus  septicaemiae  haemorrhagicae  (fowl  cholera). 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method.  The 
ends  are  commonly  more  deeply  stained  than  the  central  portion. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  yel- 
lowish-gray, finely  granular,  spherical  colonies  with  smooth  outlines  are 
developed.  In  gelatin  stick  cultures,  at  18°  to  25°  CM  at  the  end  of  twenty- 
four  hours  small,  spherical  colonies  are  developed  along  the  line  of  punc- 
ture, which  are  isolated  or  closely  crowded;  upon  the  surface  a  rather  thin, 
shining,  grayish- white,  iridescent,  circular  layer  is  formed ;  gas  is  given  off 
which  has  not  a  disagreeable  odor.  Upon  the  surface  of  agar  elevated, 
shining,  gray  colonies  develop  along  the  impfstrich,  or  a  gray,  shining  band 
is  formed  which  increases  in  thickness  but  not  in  breadth — usually  less  than 
one-half  centimetre  broad.  Old  cultures  give  off  an  acid  odor.  Upon  blood 
serum  a  thin,  white  layer  is  developed  along  the  line  of  inoculation.  Upon 
potato  a  thin,  white,  varnish- like  layer  is  formed. 

Pathogenesis.—  Very  pathogenic  for  rabbits,  guinea-pigs,  white  mice,  and 
sparrows.  Not  pathogenic  for  chickens,  pigeons,  or  geese. 


05.    BACILLUS   OF   SWINE   PLAGUE,    MARSEILLES. 

Synonyms. — Bacillus  der  Schweineseuche,    Marseilles   (Rietsch 
and  Jobert)  ;  Bacillus  der  Frettchenseuche — ferret  disease  (Eberth 


440  BACILLI  WHICH   PRODUCE   SEPTICAEMIA 

and  Schimmelbusch) ;  Bacillus  der  Amerikanischen  Rinderseuche 
(Caneva) ;  Bacillus  of  spontaneous  rabbit  septicaBmia  (Eberth). 

The  researches  of  Caneva  and  of  Bunzl-Federn  agree  as  to  the 
identity  of  the  bacillus  obtained  by  Rietsch  and  Jobert  (1887)  from 
swine  attacked  with  a  fatal  epidemic  disease  in  Marseilles,  and  the 
bacillus  found  by  Eberth  and  Schimmelbusch  (1889)  in  the  blood  of 
ferrets  suffering  from  a  fatal  form  of  septicasmia  studied  by  them. 
The  first-named  bacteriologist  also  identifies  a  bacillus  supposed 
by  Billings  to  be  the  cause  of  "Texas  fever"  in  cattle  ("  Ameri- 
kanische  Rinderseuche  ")  and  the  bacillus  of  swine  plague  (Billings) 
with  the  above.  Bunzl-Federn  obtained  cultures  of  Billings'  swine- 
plague  bacillus  at  two  different  times.  He  identifies  the  one  first  re- 
ceived with  the  bacillus  now  under  consideration,  and  the  other  with 
the  bacillus  of  hog  cholera  (Salmon).1 

Morphology. — Bacilli  with  rounded  ends,  about  twice  as  long  as 
broad,  and  one-third  smaller  than  the  bacillus  of  typhoid  fever 
(Eberth  and  Schimmelbusch).  The  bacillus  of  hog  cholera  is  shorter 
and  more  slender  than  the  Marseilles  bacillus,  and  the  bacillus  of 
Loffler  and  Schiitz  (No.  61)  is  still  smaller  (Rietsch  and  Jobert). 

In  stained  preparations  the  extremities  of  the  rods  are  usually 
deeply  stained,  while  the  central  portion  remains  unstained — "polar 
staining."  By  Loffler's  method  of  staining  the  presence  of  flagella 
may  be  demonstrated  (Frosch). 

Stains  readily  with  the  aniline  dyes  usually  employed,  but  does 
not  retain  its  color  when  treated  by  Gram's  method. 

Biological  Characters. — An  aerobic  (facultative  anaerobic), 
non-liquefying,  actively  motile  bacillus.  Grows  readily  at  the 
room  temperature,  and  is  distinguished  from  the  bacillus  of  septi- 
caemia hoemorrhagica  by  its  active  movements  and  more  rapid  and 
abundant  development  in  the  various  culture  media  usually  em- 
ployed. It  is  distinguished  from  the  bacillus  of  hog  cholera  (No.  63) 
by  producing  phenol  and  indol  in  solutions  containing  peptone,  by 
causing  coagulation  of  milk,  and  by  producing  an  acid  reaction  in 
this  fluid.  Grows  in  culture  media  having  an  acid  reaction. 

Rietsch  and  Jobert  give  the  following  account  of  the  characters 
of  growth  in  various  culture  media,  as  compared  with  the  bacillus  of 
hog  cholera  and  the  bacillus  of  Schweineseuche  (Loffler,  Schiitz), 
No.  61 : 

1  The  author  named  says :  "  With  reference  to  the  bacillus  of  swine  plague 
(Billings),  I  obtained,  as  did  Caneva,  a  decided  production  of  acid  in  the  cultures 
first  sent  by  Billings  ;  but  upon  testing  later  cultures  received  directly  from  Bil- 
lings and  from  other  sources,  the  result  was  exactly  the  opposite— viz.,  a  decided 
production  of  alkali  in  milk  and  identity  with  the  hog-cholera  bacillus  of  Salmon." 


IN   SUSCEPTIBLE   ANIMALS.  441 

Gelatin  streak  cultures.  At  the  end  of  twenty-four  hours  this 
bacillus  had  developed  considerably,  while  the  growth  of  the  hog- 
cholera  bacillus  was  scarcely  to  be  discerned  with  the  naked  eye,  and 
the  bacillus  of  Schweineseuche  did  not  form  a  visible  growth  until 
the  end  of  forty-eight  hours.  After  several  days  the  bacillus  of 
swine  plague  (Marseilles)  formed  an  opaque,  yellowish-white  streak, 
which,  when  examined  with  a  low-power  lens,  had  a  brown  color  by 
transmitted  light  and  a  bluish-white  color  by  reflected  light.  The 
streak  of  the  Lomer-Schiitz  bacillus  was  not  so  thick  and  not  so 
opaque,  and  was  made  up  of  small,  nearly  transparent  colonies  ;  the 
hog-cholera  bacillus  came  between  the  other  two.  Upon  blood 
serum,  agar,  and  glycerin-agar  the  Marseilles  bacillus  grew  more 
rapidly  than  the  other  two,  forming  a  layer  which  was  opaque  and 
of  a  white  color,  with  bluish  and  reddish  reflections.  Upon  potato 
it  formed  a  thick,  opaque,  yellowish  layer,  while  the  growth  of  the 
hog-cholera  bacillus  was  much  thinner  and  that  of  the  Loffler-Schutz 
bacillus  scarcely  to  be  seen.  In  bouillon  the  Loffler-Schutz  bacillus, 
at  the  end  of  three  days  at  37°  C.,  had  not  produced  any  perceptible 
cloudiness,  while  the  Marseilles  bacillus  at  the  end  of  twenty-four 
hours  had  caused  the  fluid  to  be  clouded,  a  film  of  bacteria  had 
formed  upon  the  surface  and  a  deposit  at  the  bottom  of  the  tube  ;  the 
hog-cholera  bacillus  produced  a  less  degree  of  opacity  in  the  bouillon. 

Pathogenesis. — This  bacillus  is  pathogenic  for  sparrows  and 
other  small  birds  when  injected  beneath  the  skin  in  small  amounts, 
and  also  for  pigeons  in  a  longer  time — five  to  fourteen  days.  Frosch 
reports  a  negative  result  from  subcutaneous  injections  into  rabbits, 
guinea-pigs,  mice,  and  pigeons,  but  his  cultures  appear  to  have  be- 
come attenuated,  as  the  recent  cultures  of  Eberth  and  Schimmelbusch 
were  fatal  to  pigeons  in  four  out  of  five  experiments.  Two  rabbits 
were  inoculated  subcutaneously  by  Rietsch  and  Jobert  with  half  a 
Pravaz  syringef  ul  of  a  pure  culture  of  the  Marseilles  bacillus ;  one  of 
these  died  on  the  sixth  day  and  the  other  survived. 

In  sparrows,  which  succumb  in  from  twenty-four  to  thirty-six 
hours  after  receiving  a  small  amount  of  a  pure  culture  in  the  breast 
muscle,  the  bacillus  is  present  in  the  blood  in  large  numbers,  and  a 
purulent  pleuritis  and  pericarditis  is  found  at  the  autopsy.  In  the 
ferrets  from  which  Eberth  and  Schimmelbusch  obtained  their  cultures 
the  bacillus  was  not  present  in  the  blood  in  sufficient  numbers  to  be 
readily  demonstrated  by  microscopical  examination,  but  it  was  ob- 
tained in  pure  cultures  from  the  liver,  spleen,  and  lungs.  The  prin- 
cipal pathological  appearances  noted  were  enlargement  of  the  spleen 
and  pneumonia.  Caneva  reports  that  the  Marseilles  bacillus  injected 
into  white  mice  gives  rise  to  an  extensive  abscess  at  the  point  of  in- 
oculation, but  does  not  kill  adult  animals.  In  a  young  mouse  which 
31 


442  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

succumbed  to  such  an  injection  the  bacilli  were  not  generally  dis- 
tributed in  the  tissues,  but  were  found  as  emboli  in  the  smaller  capil- 
laries. This  bacillus,  then,  is  distinguished  from  the  similar  bacilli 
previously  described  (Nbs.  Gl  and  63)  by  its  comparatively  slight 
pathogenic  power,  as  well  as  by  its  more  vigorous  growth  in  culture 
media,  and  the  other  characters  heretofore  mentioned. 

66.   BACILLUS  SEPTICUS  AGRIGENUS. 

Obtained  by  Nicolaier  from  soil  which  had  been  manured. 

Moi°phology.  —Resembles  the  bacillus  of  fowl  cholera  and  of  rabbit  sep- 
ticaemia, of  which  it  is  perhaps  a  variety,  but  is  usually  somewhat  longer. 
It  also  sometimes  shows  the  end-staining  characteristic  of  Bacillus  septicse- 
miae  haemorrhagicae,  but  not  so  constantly  and  not  so  sharply  defined. 

Biological  Characters.— An  aerobic,  (non  liquefying  ?),  non- motile  ba- 
cillus. Does  not  form  spores. 

In  gelatin  plate  cultures  spherical,  finely  granular  colonies  are  developed 
having  a  yellowish-brown  central  portion,  which  is  separated  by  a  dark 
ring  from  a  grayish  brown  marginal  zone;  later  this  difference  in  color  dis- 
appears and  the  colonies  become  more  decidedly  granular.  In  stick  cultures 
tne  growth  consists  of  a  thin  layer  which  is  not  at  all  characteristic. 

Pathogenesis. — Small  quantities  of  a  pure  culture  injected  into  the  ear 
vein  of  a  rabbit  cause  its  death  in  from  twenty-four  to  thirty  six  hours; 
pathogenic  also  for  house  mice  and  for  field  mice.  At  the  autopsy  no  notable 
pathological  changes  are  observed.  The  bacilli  are  found  in  blood  from  the 
neart  and  in  the  capillaries  of  the  various  organs,  but  are  not  so  numerous 
as  in  rabbit  septicaemia;  they  show  a  special  inclination  to  adhere  to  the 
margins  of  the  red  blood  corpuscles. 

67.    BACILLUS   ERYSIPELATOS   SUIS. 

Synonyms. — Bacillus  of  hog  erysipelas;  Bacillus  des  Schweine- 
rothlauf  (Loffler,  Schiitz) ;  Bacille  du  rouget  du  pore  (Pasteur)  ;  Ba- 
cillus of  mouse  septicaemia;  Bacillus  murisepticus  (Flugge) ;  Bacil- 
lus des  Mauseseptikamie  (Koch). 

The  bacillus  of  mouse  septicaemia,  first  described  by  Koch  (1878), 
resembles  so  closely  in  its  morphology,  characters  of  growth,  and 
pathogenic  power  the  bacillus  of  Schweinerothlauf  of  Loffler  and 
Schiitz  (1885)  that  they  can  scarcely  be  considered  as  distinct  spe- 
cies, although,  from  slight  differences  which  have  been  observed,  they 
are  perhaps  entitled  to  separate  consideration  as  varieties  of  the 
same  species.  Fliigge,  Eisenberg,  Frankel,  and  other  authors,  while 
recognizing  the  fact  that  the  bacilli  from  the  two  sources  closely  re- 
semble each  other,  apparently  do  not  consider  them  identical,  and 
describe  them  separately.  Baumgarten,  on  the  other  hand,  describes 
them  under  one  heading  and  considers  it  highly  probable  that  they 
are  identical,  although  he  also  admits  slight  differences  in  the 
morphological  characters  and  growth  in  culture  media.  These 
differences  are,  however,  no  greater  than  we  have  in  artificially  pro- 
duced varieties  of  other  well-known  microorganisms,  and  we  think 


IN  SUSCEPTIBLE  ANIMALS.  443 

it  best  to  follow  Baumgarten  in  describing  them  under  a  single 
heading. 

Koch  first  obtained  this  bacillus  by  injecting  putrefying  blood  or 
flesh  infusion,  during  the  first  days  of  putrefactive  change,  beneath 
the  skin  of  mice.  A  certain  proportion  of  the  animals  experimented 


FIG.  135.— Bacillus  of  mouse  septicaemia  in  leucocytes  from  blood  of  mouse,    x  700.    (Koch.) 

upon  contracted  a  fatal  form  of  septicaemia,  and  the  bacillus  under 
consideration  was  found  in  their  blood.  The  bacillus  of  Schweine- 
rothlauf  was  obtained  by  Loffler  and  by  Schiitz  from  the  blood  and 
various  organs  of  swine  which  had  succumbed  to  the  infectious 
malady  known  in  Garmany  as  rothlauf  and  in  France  as  rouget. 

Morphology. — Extremely  minute  bacilli,  about  1  /*  in  length  and 
0.2  /^  in  diameter.  The  Schweinerothlauf  bacilli  are  described  as 
somewhat  thicker  and  longer  by  Fliigge,  by  Frankel,  and  by  Eisen- 
berg,  but  Baumgarten  states  that  they  are  somewhat  more  slender  and 


Fro.  136.— Bacillus  of  rouget,  from  a  pure  culture.     X  1,000.    From  a  photomicrograph.     (Roux.) 

on  the  average  shorter  than  the  bacillus  of  mouse  septicaemia.  The 
bacilli  are  solitary,  or  in  pairs  the  elements  of  which  are  often  united 
at  an  angle;  occasionally  a  chain  of  three  or  four  elements  may  be 
observed,  and  in  old  cultures  the  bacilli  may  grow  out  into  short 


444 


BACILLI   WHICH   PRODUCE   SEPTICAEMIA 


threads  which  are  straight  or  more  or  less  curved  and  twisted.  Small 
refractive  bodies  may  sometimes  be  distinguished  in  the  rods,  and 
these  have  been  supposed  by  some  authors  to  be  spores,  but  this  has 
not  been  demonstrated. 

This  bacillus  stains  readily  with  the  ordi- 
nary aniline  staining  agents  and  also  by  Gram's 
method. 

Biological  Characters. — A  facultative  an- 
aerobic, non-liquefying  bacillus.  According  to 
Schottelius,  the  rothlauf  bacilli  are  sometimes 
motile,  but  Flugge  states  that  other  observers 
have  not  seen  them  in  active  motion.  Frankel 
says  they  have  the  power  of  voluntary  motion. 
Eisenberg  says  that  the  bacillus  of  mouse  septi- 
ca3ima  is  motionless,  and  Frankel  says  they  "  seem 
to  be  incapable  of  voluntary  motion."  Baumgar- 
ten  remarks:  "Whether  the  bacilli  exhibit  vol- 
untary movements  has  not  been  determined." 
Although  this  bacillus  is  not  strictly  anaerobic, 
it  grows  better  in  the  absence  of  oxygen  than  in 
its  presence.  Development  occurs  in  various  cul- 
ture media  at  the  room  temperature,  but  is  more 
rapid  in  the  culture  oven.  In  gelatin  stick  cul- 
tures no  development  occurs  upon  the  surface, 
but  the  growth  along  the  line  of  puncture  is  very 
characteristic;  this  consists  of  a  delicate,  cloud- 
like,  radiating  growth,  which  extends,  in  the 
course  of  a  few  days,  almost  to  the  walls  of  the  test  tube.  The 
rothlauf  bacillus  does  not  extend  so  rapidly  through  the  gelatin, 
and  the  branching,  cloud-like  growth  is  not  as  delicate;  Flugge 
compares  it  to  the  brush  of  bristles  used  for  cleansing  test  tubes. 


Fio.  187.— Bacillus  of 
mouse  septicaemia; 
culture  in  nutrient  gela- 
tin, end  of  four  days  at 
18°  C.  (Baumgarten.) 


Fio.  138.— Bacillus  of  mouse  septicaemia;  single  colony  in  nutrient  gelatin.    X  80.    (Flugge.) 

In  old  cultures  in  nutrient  gelatin  a  slight  softening  of  the  gelatin 
occurs  along  the  line  of  growth,  and  as  a  result  of  evaporation  and 
desiccation  a  funnel-shaped  cavity  is  formed  in  the  culture  medium 
in  the  course  of  two  or-three  weeks.  In  gelatin  plates  colonies  are 


IN   SUSCEPTIBLE   ANIMALS.  445 

developed  in  the  course  of  two  or  three  days  in  the  deeper  layers  of 
the  gelatin,  but  not  upon  the  surface ;  these  are  nebulous,  grayish- 
blue,  radiating  masses,  which  are  so  delicate  as  to  be  scarcely  visi- 
ble without  the  aid  of  a  lens  or  a  dark  background.  Under  a  low 
power  they  appear  as  branching  feathery  masses,  which  have  been 
compared  by  Flugge  to  the  radiating  growth  of  "bone  corpuscles," 
In  older  cultures  they  coalesce  and  cause  a  nebulous  opacity  of  the 
whole  plate,  which  has  a  bluish-gray  lustre. 

Upon  the  surface  of  nutrient  agar  or  blood  serum  a  very  scanty 
development  occurs  along  the  line  of  inoculation.  No  growth  occurs 
upon  potato.  In  bouillon  the  bacilli  cause  a  slight  cloudiness  at  the 
outset,  and  later  a  scanty,  grayish- white  deposit  upon  the  bottom  of 
the  test  tube ;  no  film  is  formed  upon  the  surface. 

The  thermal  death-point  of  this  bacillus,  as  determined  by  the 
writer  (1887),  is  58°  C.,  the  time  of  exposure  being  ten  minutes.  In 
the  experiments  of  Bolton  it  was  destroyed  in  two  hours  by  mercuric 
chloride  solution  in  the  proportion  of  1  : 10,000  ;  by  carbolic  acid  and 
by  sulphate  of  copper  in  one-per-cent  solution.  These  results  are 
opposed  to  the  view  that  the  minute  refractive  granules  which  may 
sometimes  be  seen  in  the  interior  of  the  rods  are  reproductive  spores, 
for  all  known  spores  have  a  much  greater  resisting  power  to  heat 
and  the  chemical  agents  named. 

Pathogenesis. — Pathogenic  for  swine,  rabbits,  white  mice,  house 
mice,  pigeons,  and  sparrows.  Field  mice,  guinea-pigs,  and  chickens 
are  immune. 

Swine  may  be  infected  by  the  ingestion  of  food  containing  the 
rothlauf  bacillus,  as  has  been  demonstrated  by  allowing  them  to  eat 
the  intestine  of  an  animal  which  had  recently  succumbed  to  the  dis- 
ease, and  also  by  the  subcutaneous  injection  of  pure  cultures.  The 
disease  usually  terminates  fatally  within  three  or  four  days,  and 
sometimes  in  less  than  twenty-four  hours.  It  is  characterized  by 
fever,  debility,  loss  of  appetite,  and  by  the  appearance  upon  the  sur- 
face of  the  body  of  red  patches,  which  gradually  extend  and  become 
confluent,  producing  after  a  time  a  uniform  dark-red  or  brown  color 
of  the  entire  surface.  The  discharges  from  the  bowels  frequently 
contain  bloody  mucus.  At  the  autopsy,  in  acute  cases,  the  spleen  is 
notably  enlarged,  and  the  liver  and  kidneys  are  likely  to  be  more  or 
less  swollen,  as  are  also  the  lymphatic  glands,  especially  those  of 
the  mesentery;  the  gastric  and  intestinal  mucous  membranes  are 
usually  inflamed  and  spotted  with  hsemorrhagic  extravasations  ;  the 
serous  membranes  also  may  be  inflamed,  and  the  cavities  of  the 
pleura3,  pericardium,  and  peritoneum  usually  contain  more  or  less 
fluid.  The  bacilli  are  found  in  the  blood  vessels  throughout  the 


446  BACILLI  WHICH   PRODUCE  SEPTICAEMIA 

body  and  are  especially  numerous  in  the  interior  of  the  leucocytes. 
Cornevin  and  Kitt  have  shown  that  the  contents  of  the  intestine 
also  contain  the  bacilli  in  large  numbers,  and  the  disease  appears  to 
be  propagated  among  swine  principally  by  the  contamination  of  their 
food  with  the  alvine  discharges  of  diseased  animals. 

Pigeons  are  very  susceptible  to  the  pathogenic  action  of  this  ba- 
cillus, and  usually  die  within  three  or  four  days  after  inoculation 
with  a  pure  culture.  Rabbits  are  not  so  susceptible,  although  a 
certain  proportion  die  from  general  infection  after  being  inoculated 
in  the  ear.  The  first  effect  of  such  an  inoculation  is  to  produce  an 
erysipelatous  inflammation.  When  the  animal  recovers  it  is  subse- 
quently immune. 

White  mice  and  house  mice  are  extremely  susceptible,  but  field 


Fro.  189.— Section  of  dlaphrasrm  of  a  mouse  dead  from  mouse  septicaemia,  showing  bacilli  in  a 
capillary  blood  vessel.    (Baumgarten.) 

mice  are  immune.  This  remarkable  fact  was  first  ascertained  by 
Koch  by  experiments  with  his  bacillus  of  mouse  septicaemia.  House 
mice  which  have  been  inoculated  with  a  minute  quantity  of  a  pure 
culture  of  the  rothlauf ,  or  mouse  septicaemia,  bacillus,  die  in  from 
forty  to  sixty  hours.  The  animal  is  usually  found  dead  in  a  sitting 
position,  with  its  back  strongly  curved,  and  for  many  hours  before 
death  it  remains  quietly  sitting  in  the  same  position  ;  the  eyes  are 
glued  together  by  a  sticky  secretion  from  the  conjunctival  mucous 
membrane.  At  the  autopsy  the  spleen  is  found  to  be  very  much  en- 
larged, and  there  may  be  a  slight  amount  of  oedema  at  the  point  of 
inoculation. 

The  bacilli  are  found  in  the  blood  vessels  generally,  and  are  very 


IN  SUSCEPTIBLE  ANIMALS.  447 

numerous  in  the  interior  of  the  leucocytes,  which  are  sometimes  com- 
pletely filled  with  them. 

Pasteur's  first  studies  relating  to  the  etiology  of  "rouget"  were 
made,  in  collaboration  with  Chamberlain,  Roux,  and  Thuillier,  in 
1882.  His  description  of  the  microorganism  to  which  he  attributed 
the  disease  does  not  correspond  with  that  subsequently  isolated  by 
Loffler  an  I  by  Schiitz  ;  but  the  last-named  bacteriologists,  and  Schot- 
telius  also,  found  the  characteristic  rothlauf  bacillus  in  cultures  from 
his  laboratory  which  had  baan  prepared  for  the  protective  inoculation 
of  swine — "  vaccins."  Pasteur  found,  by  experimental  inoculations 
of  his  bacillus  of  rouget  into  pigeons,  that  the  virulence  of  his  cul- 
tures was  increased  by  successive  inoculations  through  a  series  of 
these  birds,  as  shown  by  the  oocurrenca  of  daath  at  an  earlier  date, 
and  also  by  the  fact  that  blood  taken  from  the  last  pigeon  in  a  series 
was  more  virulent  for  swine  than  that  from  the  first  or  from  an  in- 
fected pig.  On  the  other  hand,  the  virulence  was  diminished  by  in- 
oculations into  rabbits  ;  and,  by  passing  the  bacillus  through  a  series 
of  these  animals,  a  vaccine  was  obtained  which  produced  a  com- 
paratively mild  and  non-fatal  attack  in  swine.  In  practice  the  U33 
of  two  different  vaccines  is  recommended,  a  mild — "  attenuated " 
—virus  being  first  inoculated,  and,  after  an  interval  of  twelve  days, 
a  second  vaccine  having  greater  pathogenic  potency.  These  inocula- 
tions have  been  extensively  practised  in  France,  and  that  immunity 
from  the  disease  may  be  secured  in  this  way  is  well  established,  hav- 
ing been  confirmed  in  Germany  by  Schiitz,  by  Lydtin,  and  by  Schot- 
telius.  There  is,  however,  some  doubt  as  to  the  practical  value  of 
the  method,  inasmuch  as  a  certain  number  of  the  inoculated  animals 
die,  and  there  appears  to  be  danger  that  the  disease  may  be  spread 
by  the  alvine  discharges  of  inoculated  animals.  In  a  region  where 
the  annual  losses  from  tho  disease  are  considerable,  and  where  the 
soil  is,  perhaps,  thoroughly  infected  with  rothlauf  bacilli,  protective 
inoculations  probably  afford  the  best  security  against  loss.  But 
under  other  circumstances  the  quarantine  of  infected  animals  and 
thorough  disinfection  of  the  localities  in  which  cases  have  occurred 
will  probably  prove  a  better  mode  of  procedure. 

68.    BACILLUS  COPROGENES  PAKVUS. 

Synonym.—  Mauseseptikamieahnlicher  Bacillus  (Eisenberg). 

Obtained  by  Bienstock  from  human  faeces. 

Morphology. — A  very  minute  bacillus,  which  is  but  little  longer  than  it 
is  broad,  and  might  easily  be  mistaken  for  a  raicrococcus. 

Biological  Characters.— Grows  very  slowly  on  nutrient  gelatin,  forming 
a  scarcely  visible  film  along  the  line  of  inoculation,  which  at  the  end  of 
several  weeks  is  scarcely  one  millimetre  wide.  Is  not  motile. 

Pathogenesis. — In  white  mice  an  extensive  O3dema  is  developed  at  the 


448  BACILLI   WHICH   PRODUCE   SEPTICAEMIA. 

point  of  inoculation  at  the  end  of  ten  or  twelve  hours,  and  the  animal  dies 
within  thirty-six  hours.  The  bacilli  are  found  in  great  numbers  in  the 
effused  serum  at  the  point  of  inoculation  and  in  comparatively  small  num- 
bers in  the  blood.  A  rabbit  inoculated  with  a  pure  culture  obtained  from  a 
mouse  died  at  the  end  of  eight  days.  The  inoculation,  which  was  made  in 
the  ear,  gave  rise  to  a  local  erysipelatous  inflammation. 

69.    BACILLUS  CAVICIDA. 

Synonym.—  Brieger's  bacillus.  Probably  a  pathogenic  variety  of  Bac- 
terium coli  commune  of  Escherich. 

Obtained  by  Brieger  (1884)  from  human  fasces. 

Morphology — Small  bacilli,  about  twice  as  long  as  broad,  which  closely 
resemble  the  colon  bacillus  of  Escherich  (Bacterium  coli  commune). 

Biological  Characters. — An  aerobic  (facultative  anaerobic),  non-liquefy- 
ing bacillus. 

The  growth  in  gelatin  plate  cultures  is  said  to  be  very  characteristic,  the 
colonies  being  "  in  the  form  of  very  beautifully  grouped,  whitish,  concentric 
rings,  which  are  arranged  like  the  ccales  upon  the  back  of  a  turtle"  (Eisen- 
bergi.  The  writer  has  studied  cultures  of  this  bacillus  brought  from  the 
bacteriological  laboratories  of  Germany,  side  by  side  with  cultures  of  the 
Bacterium  coli  commune  of  Escherich,  and  has  found  no  appreciable  differ- 
ences in  the  colonies  in  gelatin  plates,  or  in  the  growth  in  various  culture 
media.  Upon  potato  it  grows  rapidly  in  the  incubating  oven,  forming  a 
dirty-yellow,  moist  layer. 

Pathogenesis. — This  bacillus,  as  first  obtained  by  Brieger,  was  character- 
ized by  being  very  pathogenic  for  guinea-pigs, which  were  invariably  killed, 
within  seventy-two  hours,  by  the  subcutaneous  injection  of  a  minute  quan- 
tity of  a  pure  culture.  The  bacillus  was  found  in  great  numbers  in  the 
blood  of  animals  which  succumbed  to  an  experimental  inoculation.  The 
writer's  experiments  with  this  bacillus,  made  in  1889,  indicate  that  its  patho- 
genic power  had  become  attenuated,  inasmuch  as  considerable  quantities  of 
a  pure  culture  injected  into  guinea-pigs  did  not  cause  the  death  of  the  ani- 
mals— culture  used  came  originally  from  Germany.  Not  pathogenic  for 
rabbits  or  for  mice. 

70.    BACILLUS   CAVICIPA   HAVANIENSIS. 

This  bacillus  was  obtained  by  the  writer  from  the  contents  of  the  intestine 
of  a  yellow-fever  cadaver,  in  Havana,  1889,  through  inoculated  guinea-pigs. 

Morphology. — A  bacillus  with  rounded  ends, 
from  two  to  three  p  long  and  about  0.7  //  broad, 
frequently  united  in  pairs. 

Stains  readily  with  the  ordinary  aniline  colors. 
Biological  Characters. — An  aerobic  and  fac- 
ultative anaerobic,  non -liquefying,  actively  mo- 
tile bacillus. 

In  gelatin  stick  cultures  the  growth  upon  the 
surface  is  very  scanty  and  thin,  not  extending  far 
from  the  point  of  puncture  ;  along  the  line  of 
puncture  are  developed  small,  translucent,  pearl- 
like,  spherical  colonies,  which  later  become  opaque 
and  sometimes  granular.     In  gelatin  roll  tubes, 
Fio.  i40.-Baciiius  cavicida   at   the  end  of    twenty-four    hours    at   22°    C., 
Havaniensis;  from   a  potato   the  deep  colonies  are  very  small  spheres,  of  a  pale 
culture,    x  1,000.  Fromapho-   straw  color ;  later  they  become  opaque,  light  brown 
tomicrograph.   (Sternberg.)      spheres,  or  may  have  a  dark  central  mass  sur- 
rounded by  a  transparent  zone.    The  superficial 

colonies  at  the  end  of  five  days  are  small,  translucent  masses  of  a  pale  straw 
color  towards  the  centre,  with  thin  and  irregular  margins,  sometimes  with 


IN  SUSCEPTIBLE   ANIMALS. 


449 


a  central  light-brown  nucleus ;  at  the  end  of  ten  days  the  deep  colonies  are 
still  quite  small,  of  a  brown  color,  and  opaque. 

In  glycerin-agar  roll  tubes,  at  the  end  of  twenty-four  hours,  the  deep  colo- 
nies are  in  the  form  of  a  Biconvex  lens,  and  appear  spherical  when  viewed 
in  face  and  biconvex  when  seen  from  the  side ;  they  have  a  straw  color 
by  transmitted  light  and  are  bluish- white  by  reflected  light ;  the  superficial 
colonies  are  translucent,  with  a  bluish  white  lustre, 

On  potato,  at  22°  C.,  at  the  end  of  forty  eight  hours  there  is  a  thin,  dirty- 
yellow  growth  of  limited  extent;  at  the  end  of  ten  days  there  is  a  thin, 
gamboge  yellow  layer  and  little  masses  of  the  same  color;  the  growth  is 
quite  thin,  with  irregular  outlines,  and  is  confined  to  the  vicinity  of  the 
impfstrich.  \ 

Grows  in  nutrient  agar  containing  0.2  per  cent  of  hydrochloric  acid. 
Thermal  death  point  55°  C.  Grows  in  agua  coco  without  forming  gas,  and 
causes  this  liquid  and  bouillon  to  become  slightly  translucent— not  milky. 

Pathogenesis. — Pathogenic  for  guinea-pigs,  less  so  for  rabbits.  Guinea- 
pigs  inoculated  subcutaneously  with  a  few  drops  of  a  pure  culture  die  in  ten 
or  twelve  hours  from  general  infection.  There  is  usually  a  considerable 
effusion  of  bloody  serum  in  the  vicinity  of  the  point  of  inoculation,  and  the 
spleen  is  more  or  less  enlarged. 

71.  BACILLUS  CRASSUS  SPUTIGENUS, 

Obtained  by  Kreibohm  (1886)  from  the  sputum  of  two  individuals,  and 
once  in  scrapings  from  the  tongue. 


Morphology. — Short,  thick  bacilli,  of  oblong  form,  with  rounded  corners, 
en  bent  or  twisted — "sausage-shaped."    Immediately  after  division  the 
bacilli  are  about  one-half  longer  than  they  are  broad,  but  before  dividing 


FIG.  141.— Bacillus  crassus  sputigenus,  from  blood  of  mouse,    x  700.    (Flug^e.) 

again  they  may  attain  a  length  of  three  to  four  times  the  breadth.    Irregular 
forms  with  swollen  ends  or  uneven  contour  are  frequently  seen. 

This  bacillus  is  quickly  stained  by  the  ordinary  aniline  colors  and  also 
by  Gram's  method. 

Biological  Characters.— An  aerobic,  non-liquefying  (non-motile  ?)  ba- 
cillus.    Grows  in  various  culture  media  at  the  room  temperature— more 
rapidly  in  the  incubating  oven.    "Appears  to  form   spores   at   65    U. 
(Fliigge). 

In  gelatin  plates,  at  the  end  of  thirty-six  hours,  grayish- white  colonies  are 
developed,  which  soon  reach  the  surface  of  the  gelatin  and   spread  out  as 


450  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

round,  viscid,  grayish- white  drops,  which  project  considerably  above  the 
surface  of  the  culture  medium.  Under  a  low  magnifying  power  recent  colo- 
nies appear  as  spherical,  grayish -brown  discs,  the  surface  of  which  is  marked 
with  dark  points  or  lines.  The  superficial  colonies  are  more  transparent, 
have  irregular  outlines,  and  the  surface,  especially  near  the  margins,  is 
coarsely  granular.  The  development  in  stick  cultures  is  very  rapid  and  re- 
sembles that  of  Friedlander's  bacillus — "  nail-shaped  "  growth.  Upon  potato 
the  growth  is  also  similar  to  that  of  Friedlander's  bacillus,  and  consists  of  a 
thick,  grayish-white,  moist,  and  shining  layer. 

Pathogenesis.—yLice  inoculated  with  a  small  quantity  of  a  pure  culture 
die  from  acute  septicaemia  in  about  forty-ei^ht  hours.  The  bacilli  are  found 
in  blood  from  the  heart  and  from  the  various  organs — most  numerous  in 
the  liver.  Rabbits  are  killed  within  forty  eight  hours  by  intravenous  injec- 
tion of  a  small  quantity,  and  the  blood  contains  the  bacillus  in  great  num- 
bers. Larger  amounts  injected  into  the  circulation  of  rabbits  or  dogs  cause 
death  in  a  few  hours  (three  to  ten),  preceded  by  diarrhoea,  and  in  some  in- 
stances bloody  discharges  from  the  bowels.  At  the  autopsy  an  acute  gastro- 
enteritis is  found. 

72.    BACILLUS  PYOGENES  FCETIDUS. 

Obtained  by  Passet  (1885)  from  an  abscess  of  the  anus. 

Morphology.—  Short  bacilli  with  rounded  ends,  1.45  jn  long  and  0.58  n 
broad ;  usually  associated  in  pairs  or  in  short  chains. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Grows  rapidly  in  the  usual  culture  media  at  the  room  temperature.  In  the 
interior  of  the  rods,  in  stained  preparations,  one  or  two  unstained,  spherical 
places  may  sometimes  be  seen,  which  have  been  supposed  to  be  spores  (?). 
The  independent  motion  exhibited  by  this  bacillus  is  not  very  active.  In 
gelatin  plates  white  colonies  are  developed  at  the  end  of  twenty-four  hours, 
which  upon  the  surface  spread  out  as  grayish-white  plaques,  having  a  dia- 
meter sometimes  of  one  centimetre ;  these  are  thickest  in  the  centre  and  of 
a  whitish  color ;  the  colonies  may  become  confluent.  In  gelatin  stick  cul- 
tures the  growth  upon  the  surface,  at  the  end  of  twenty-four  hours,  consists 
of  a  thin,  grayish- white  layer  with  rather  thick,  irregular  margins;  along  the 
line  of  puncture  more  or  less  crowded  colonies.  Upon  potato >the  bacillus 
forms  an  abundant,  shining,  pale-brown  layer.  The  cultures  give  off  a  dis- 
agreeable putrefactive  odor. 

According  to  Eisenberg,  mice  and  guinea-pigs  are  killed  in  twenty-four 
hours  by  injections  beneath  the  skin  or  into  the  cavity  of  the  abdomen,  and 
numerous  bacilli  are  found  in  the  blood. 

73.  PROTEUS  HOMINIS  CAPSULATUS. 

Obtained  by  Bordoni-Uffreduzzi  (1887)  from  two  cadavers  presenting  the 
pathological  appearances  of  the  so-called  "  Hadernkrankheit." 

Morphology. — Bacilli,  varying  considerably  in  dimensions;  somewhat 
thicker  than  the  anthrax  bacillus;  often  swollen  in  the  middle  or  at  the  ex- 
tremities: more  or  less  curved;  isolated,  united  in  pairs  or  in  long  filaments; 
in  stained  preparations  from  agar  cultures  or  from  blood  the  bacilli  are  sur- 
rounded by  a  "capsule." 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  (facultative  anaerobic  ?),  non-lique- 
jying*  non-motile  bacillus.  Formation  of  spores  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  At  a  temperature  of  15°  to 
17°  C.  long  filaments  are  formed,  in  which  the  bacilli  are  surrounded  with  a 
rapsulo;  at  22  to  24 "  C.  the  bacilli  are  for  the  most  part  isolated,  but  few  fila- 
ments being  formed  ;  at  32°  to  37°  C.  the  bacilli  are  so  short  as  to  resemble 
micrococci;  development  ceases  at  a  temperature  of  8°  and  is  very  slow  at 
16  C. 


IN   SUSCEPTIBLE   ANIMALS. 


451 


Fro.  148,— Proteus  hominis  capsulatus,  from 
liver  of  mouse.  X  1,000.  (Bordoni.Uffre- 
duzzi.) 


This  bacillus  grows  as  well  m  an  acid  medium  as  in  one  which  is  slightly 
alkaline.     In  gelatin  plates,  at  the  end  of  eighteen  to  twenty-four  hour/ 
colonies  are  formed  which  under  a  low  power  are  seen  to  be  spherical  and 
to  contain  a  quantity  of  shining  granules;  the  following  day,  at  a  tempera- 
ture ot  15   to  17    C.,  the  colonies  may  be  as  large  as  a  phi's  head  and  still 
remain  spnerical  or  slightly  oval,  but 
the  outline  is  no  longer  so  uniform, 
and  between  the  shining  points  in  the 
interior  a  confused  network  may  be 
seen ;  as  the  colony  becomes  larger  it 
is  raised  above  the  surface  of  the  gela- 
tin, becomes  opaque,  and  has  a  pearly 
lustre  like  that  of  Friedlander's  bacil- 
lus.   In   gelatin    stick   cultures   the 
growth  resembles   that  of    Friedlan- 
der's bacillus — "  nail-shaped  growth." 
Upon   the  surface  of  nutrient  agar  a 
rapidly  extending,    semi  transparent 
layer  is  formed.     Upon  potato,  at  15° 
to  17°  C.,  at  the  end  of  twenty-four 
hours  transparent  drops  are  seen  in 
the  vicinity  of  the  point  of  inocula- 
tion, and  later  a  moist,  shining,  color- 
less layer,   of  tough    consistence,   is 
formed,  which  gradually  extends  over 
the  surface.     The  growth  upon  blood 
serum  resembles  that  upon  nutrient 
agar,  and  the  blood  serum  is  not  liquefied'.    In  liquid  blood  serum  or  in 
bouillon  the  bacilli  are  isolated — not  in  filaments ;  they  cause  a  clouding  of 
the  liquid,  and  an  abundant  deposit  accumulates  at  the  bottom  of  the  tube, 
while  a  film  of  bacilli  forms  upon  the  surface.     The  cultures  never  give  off 
a  putrefactive  odor. 

Pathogenesis.— Pathogenic  for  dogs  and  for  mice,  less  so  for  rabbits  and 
for  guinea-pigs.  Agar  cultures  grown  in  the  incubating  oven  at  32°  to  37 
C.  are  more  pathogenic  than  cultures  in  gelatin  at  the  room  temperature. 
A  small  quantity  of  a  recent  culture  injected  subcutaneously  in  mice  causes 
their  death  in  from  one  to  four  days,  according  to  the  quantity  and  age  of 
the  culture;  the  recent  cultures  are  most  virulent.  When  the  animal  lives 
more  than  twenty -four  hours  it  has  a  mucous  diarrhoea.  At  the  autopsy  the 
spleen  is  found  to  be  much  enlarged  and  dark  in  color  ;  the  lymphatic 
glands  are  also  swollen  and  haemorrhagic,  the  liver  and  kidneys  hyperaemic; 
in  the  vicinity  of  the  point  of  inoculation  is  a  subcutaneous  cedema  of  jelly- 
like  appearance  and  numerous  punctiform  haemorrhages  are  seen.  The  ba- 
cillus is  found  in  great  numbers  in  the  effused  serum  from  the  subcutaneous 
tissues,  in  the  blood,  the  contents  of  the  intestine,  and  in  the  parenchyma  of 
the  various  organs.  When  examined  at  once  the  bacilli  in  the  subcutaneous 
oedema  and  in  the  lymphatic  glands  are  usually  quite  short,  and  even  spherical, 
while  in  the  blood  they  are  somewhat  longer  and  may  appear  as  short  fila- 
ments with  swollen  ends,  surrounded  by  a  capsule.  When  the  examination 
is  made  some  time  after  the  death  of  the  animal  longer  filaments  are  quite 
numerous.  Rabbits  and  guinea-pigs  are  killed  by  the  intravenous  injection 
of  comparatively  small  amounts  of  a  recent  culture,  but  quite  large  doses 
are  required  to  produce  a  fatal  result  when  the  injection  is  made  beneath 
the  skin.  From  two  to  three  cubic  centimetres  of  a  recent  culture  injected 
into  the  circulation  of  a  dog  give  rise  to  symptoms  of  toxaemia,  and  the  ani- 
mal usually  dies  on  the  second  day.  At  the  autopsy  the  abdominal  organs 
are  found  to  be  hyperaemic,  the  mucous  membrane  of  the  intestine  swollen, 
red  in  color,  and  covered  with  bloody  mucus.  The  bacillus  is  found  in  the 
blood  and  in  the  various  organs.  When  smaller  doses  are  injected  into  a 
vein  (a  few  drops)  the  animal,  after  a  few  hours,  has  a  mucous  diarrhoea  and 


452  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

vomiting,  or  efforts  to  vomit.  Death  usually  occurs  at  the  end  of  two  or 
three  days.  At  the  autopsy  the  spleen  is  found  to  be  normal,  the  other  or- 
gans slightly  hyperaemic,  and  the  intestinal  mucous  membrane  in  a  state  of 
catarrhal  inflammation.  The  bacilli  are  found  in  the  blood  and  in  the  vari- 
ous organs  in  considerable  numbers. 

74.    PROTEUS  CAPSULATUS   SEPTICUS. 

Obtained  by  Banti  (1888)  from  a  case  of  "  acute  hsemorrhagic  infection/' 
According  to  Banti,  this  is  possibly  identical  with  the  preceding  species- 
Proteus  hominis  capsulatus — but  in  some  respects  more  nearly  resembles 
Friedlander's  bacillus. 

75.    BACILLUS  ENTERITIDIS. 

Obtained  by  Gartner  (1888)  from  the  tissues  of  a  cow  which  was  killed  in 
consequence  of  an  attack  charactei  ized  by  a  mucous  diarrhoea,  and  also  from 
the  spleen  of  a  man  who  died  twelve  hours  after  eating  the  flesh  of  this 
animal. 

Morphology. — Short  bacilli,  about  twice  as  long  as  broad,  frequently  united 
in  pairs;  chains  of  four  to  six  elements  are  sometimes  seen. 

Stains  with  the  usual  aniline  colors,  and  presents  the  peculiarity  of 
staining  deeply  at  one  end  while  the  remainder  of  the  rod  is  but  slightly 
stained.  When  two  bacilli  are  united  the  deeply  stained  ends  are  in  apposi- 
tion. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Spore  formation  not  determined.  Grows  in  the  usual  culture  media  at  the 
room  temperature.  Upon  gelatin  plates  pale-gray,  superficial  colonies  are 
formed  at  the  end  of  twenty-four  hours;  under  a  low  power  these  are  seen 
to  be  coarsely  granular  and  transparent;  the  central  portion  usually  pre- 
sents a  greenish  color;  deep  colonies  are  spherical,  indistinctly  granular, 
and  of  a  brownish  color  ;  in  older  colonies  a  marginal  transparent  zone  is 
seen  which  appears  to  be  made  up  of  minute  fragments  of  glass  of  a  pale- 
brown  color.  In  gelatin  stick  cultures  but  slight  development  occurs  along 
the  line  of  puncture  ;  upon  the  surface  a  thick,  grayish-white  layer  is 
formed,  which  after  a  time  becomes  very  much  wrinkled.  Upon  the  surface 
of  agar,  at  37°  C.,  at  the  end  of  eighteen  to  twenty 'hours  a  grayish- yellow 
layer  has  formed.  Upon  potato  a  moist,  shining,  yellowish-gray  layer  is 
developed.  The  growth  upon  blood  serum  is  rapid  in  the  form  of  a  gray 
layer  along  the  line  of  inoculation. 

Pathogenesis. — White  mice  and  house  mice  usually  die  in  from  one  to 
three  days  when  fed  with  a  pure  culture  of  this  bacillus.  Rabbits  and  gui- 
nea-pigs die  in  from  two  to  five  days  from  subcutaneous  injections— less 
pathogenic  for  pigeons  and  canary  birds.  Dogs,  cats,  chickens,  and  sparrows 
are  immune.  A.  goat  died  in  twenty  hours  after  receiving  an  intravenous 
injection  of  two  cubic  centimetres  or  a  culture  in  blood  serum.  The  princi- 
pal pathological  appearance  consists  in  an  intense  inflammation  of  the  in- 
testinal mucous  membrane.  The  bacilli  are  found  in  blood  from  the  heart 
and  also  in  the  contents  of  the  stomach. 

76.    BACILLUS  OF  GROUSE  DISEASE. 

Obtained  by  Klein  (1889)  from  the  lungs  and  liver  of  grouse  which  had 
succumbed  to  an  epidemic  disease. 

Morphology.—  Bacilli  with  rounded  ends,  from  0.8  to  1.6 //long;  may 
also  be  seen  as  spherical  or  oval  cells  0.6  //  long  and  0.4  /*  thick;  solitary,  in 
pairs,  or  in  chains  of  three  to  four  elements. 

Stains  best  with  Weigert's  solution  of  methylene  blue  in  aniline  water. 

Biological  Characters  —  An  aerobic,  non-liquefying,  non-motile  bacillus. 
Spore  formation  not  observed.  Grows  in  the  usual  culture  media  at  the 


IN   SUSCEPTIBLE   ANIMALS.  453 

room  temperature — better  in  the  incubating  oven.  Upon  gelatin  plates,  at 
20°  C.,  at  the  end  of  twenty-four  hours  small,  angular,  transparent  scales 
may  be  seen  upon  the  surface  with  a  low- power  lens;  at  the  end  of  three  or 
four  days  these  form  flat,  more  or  less  irregular,  shining,  gray  colonies,  with 
thin  and  of  ten  dentate  margins ;  these  colonies  may  become  confluent  and 
form  a  dry,  scaly  layer  which  by  reflected  light  has  a  peculiar,  fatty  lustre 
In  gelatin  stick  cultures  the  superficial  growth  is  in  the  form  of  a  trans- 
parent, dry,  grayish  layer  with  dentate  margins,  not  more  than  three  to  five 
millimetres  in  diameter.  Upon  agar,  at  36°  to  37°  C.,  a  thin,  whitish-gray, 
dry  layer  is  formed. 

Pathogenesis. — Pathogenic  for  mice,  for  guinea-pigs,  for  linnets,  and  for 
green-finches;  less  so  for  sparrows.  Chickens,  pigeons,  and  rabbits,  accord- 
ing to  Klein,  are  immune.  Of  eight  mice  inoculated  subcutaneously  with 
one  or  two  drops  of  a  bouillon  culture,  six  died  within  forty- eight  hours 
and  two  recovered.  Out  of  eight  guinea-pigs  inoculated  in  the  same  way 
four  died  in  forty-eight  hours  and  two  recovered.  At  the  autopsy  the 
lungs  and  liver  were  found  to  be  hyperaemic,  the  spleen  not  enlarged.  The 
bacilli  were  present  in  large  numbers  in  blood  from  the  heart  and  in  the 
lungs. 

77.   BACILLUS  GALLINARUM. 

Obtained  by  Klein  (1889)  from  the  blood  of  chickens  which  succumbed 
to  an  epidemic  disease  resembling  u  fowl  cholera."  The  bacillus  is  believed 
by  Klein  not  to  be  identical  with  Pasteur's  bacillus  of  fowl  cholera,  and  is 
said  not  to  be  pathogenic  for  rabbits,  which  would  seem  to  differentiate  it 
from  this  bacillus  ( Bacillus  septicaemias  haemorrhagicae). 

Morphology. — Bacilli  with  rounded  ends,  from  0.8  to  2  n  long  and 
0.3  to  0.4  u  thick ;  often  in  pairs. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motile  bacillus. 
Does  not  form  spores.  Grows  in  the  usual  culture  media  at  the  room  tem- 
perature—better in  the  incubating  oven.  Upon  gelatin  plates  forms  grayish- 
white,  superficial  colonies,  which  later  present  the  appearance  of  flat,  homo- 
geneous, whitish  discs  with  thin  edges  and  irregular  margins,  and  by 
transmitted  light  have  a  brownish  color.  The  deep  colonies  are  small  and 
spherical,  and  have  a  brownish  color  by  transmitted  light.  In  gelatin  stick 
cultures  a  thin,  gray  layer  with  irregular  margins  and  of  limited  extent 
forms  upon  the  surface,  and  a  scanty  growth  occurs  along  the  line  of  punc- 
ture in  the  form  of  a  grayish- white  line.  Upon  the  surface  of  agar,  at 
37°  C.,  a  thin,  gray  layer  with  irregular  margins  has  developed  at  the  end  of 
twenty-four  hours ;  later  this  extends  over  the  entire  surface  as  a  thin,  gray- 
ish-white layer.  No  growth  occurs  upon  potato  at  37°  C.  In  bouillon,  at  37° 
C.,  development  occurs,  with  clouding  of  the  bouillon,  within  twenty -four 
hours;  later  a  deposit  consisting  of  bacilli  is  seen  at  the  bottom  of  the  tube, 
but  no  film  forms  upon  the  surface. 

Pathogenesis.  —Chickens  inoculated  subcutaneously  with  a  pure  culture 
die  in  from  twenty-four  hours  to  eight  or  nine  days.  Pigeons  and  rabbits 
are  immune. 

78.    BACILLUS   SMARAGDINUS   FCETIDUS. 

Obtained  by  Reimann  (1887)  from  the  nasal  secretions  in  a  case  of 
ozaena. 

Morphology. — Small,  slender,  slightly  curved  bacilli,  about  half  as  large 
as  the  tubercle  bacilli;  usually  arranged  in  parallel  groups. 

Biological  Characters.— Anaerobic  and  facultative  anaerobic,  liquefying 
bacillus.  Spore  formation  not  observed.  Grows  slowly  at  the  room  tem- 
perature in  the  usual  culture  media— more  rapidly  at  37°  C.  In  gelatin  stick 
cultures  development  occurs  along  tha  line  of  puncture,  and  at  the  end  of 
forty-eight  hours  a  slight  liquefaction,  in  form  of  a  funnel,  occurs  near  the 


454  BACILLI   WHICH   PRODUCE    SEPTICAEMIA 

surface ;  after  the  eighth  day  liquefaction  progresses  more  rapidly.  About 
the  sixth  day  a  bright-green  color  is  recognized  in  the  upper  part  of  the  tube 
by  reflected  light.  Upon  agar  plates,  at  37°  C.,  at  the  end  of  forty-eight 
hours  minute  colonies  are  formed,  of  irregular  form,  which  have  a  white 
color  with  a  shade  of  green ;  in  older  colonies  the  central  portion  may  be 
finely  granular  and  brownish  yellow  in  color,  while  the  marginal  zone  is 
more  transparent;  the  agar  has  by  reflected  light  a  deep  emerald  green  color. 
In  agar  stick  cidtures,  at  the  end  of  twenty  hours,  an  abundant  development 
has  occurred  without  color;  at  the  end  of  forty-eight  hours  the  culture  me- 
dium is  of  a  bright  green  color  throughout ;  later  the  color  changes  to  brown. 
A  dirty-yellow  layer  forms  upon  the  surface  of  the  agar.  Upon  potato,  at 
37°  C.,  a  dark- brown  layer  forms  in  the  vicinity  of  the  line  of  inoculation; 
later  this  is  chocolate  brown. 

The  cultures  in  gelatin  and  agar  give  off  a  peculiar,  penetrating  odor 
similar  to  that  of  jasmin. 

Pathogenesis.  —  Pathogenic  for  rabbits  when  injected  into  a  vein  or  sub- 
cutaneously.  Death  occurs  in  from  thirty  six  to  forty- eight  hours.  At  the 
autopsy  haemorrhagic  extravasations  are  found  beneath  the  pericardium  and 
the  pleurae;  abscesses  in  the  lungs  and  liver.  The  bacilli  are  found  in  the 
blood  and  in  the  various  organs  in  large  numbers. 

79.   BACILLUS  PNEUMOSEPTICUS. 

Obtained  by  Babes  (1889)  from  the  blood  and  tissues  of  an  individual  who 
died  of  septic  pneumonia. 

Morphology.  —  Small,  straight  bacilli  about  0.2 //  thick. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, non-motile  bacillus.  Spore  formation  not  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  superfi- 
cial colonies  are  formed  which  are  flat,  irregular  in  outline,  whitish,  shining, 
and  semi  transparent ;  under  a  low  power  finger  like  offshoots  are  seen  about 
the  periphery.  In  gelatin  stick  cultures  an  abundant  development  occurs 
along  the  line  of  puncture;  the  colonies  give  off  a  strong  sperm-like  odor. 
Upon  the  surface  of  agar  small,  whitish,  flat,  shining  colonies  with  ill  de- 
fined outlines  are  formed,  which  soon  become  confluent  arid  cover  the  sur- 
face; an  abundant  white  deposit  is  seen  in  the  condensation  water.  Upon 
potato  a  moist,  white  layer  is  formed.  Upon  blood  serum  circular,  whitish, 
transparent  colonies  are  formed  along  the  line  of  inoculation,  which  soon 
coalesce. 

Pathogenesis. — Very  pathogenic  for  rabbits,  guinea-pigs,  and  mice  when 
injected  subcutaneously  in  small  amount.  The  animals  die  in  from  two  to 
three  days  without  any  noticeable  local  inflammation  and  with  symptoms  of 
septicaemia.  The  lungs  and  spleen  are  found  to  be  hyperaemic.  The  bacilli 
are  found  in  the  blood  free,  or  sometimes  enclosed  in  the  leucocytes;  they 
are  only  found  in  small  numbers  in  the  capillaries  of  the  internal  organs. 
Cultures  gradually  lose  their  virulence  when  propagated  in  artificial  media. 

80.   BACILLUS  CAPSULATU8. 

Obtained  by  Pfeiffer  (1889)  from  the  blood  of  a  guinea  pig  which  died 
spontaneously. 

Morphology.—  Thick  bacilli  with  rounded  ends,  usually  two  or  three 
times  as  long  as  broad;  often  united  in  chains  of  two  or  three  elements;  may 
prow  out  into  homogeneous  filaments.  Stained  preparations  show  the  ba- 
cilli to  be  enveloped  in  an  oval  capsule  which  may  be  considerably  broader 
than  the  bacilli  themselves— two  to  five  times  as  broad ;  where  several  ba- 
cilli are  united  they  are  surrounded  by  a  single  capsular  envelope. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method.  In  pre- 
parations which  are  deeply  stained  with  hot  f  uchsin  or  gentian  violet  solu- 


IN   SUSCEPTIBLE   ANIMALS.  455 

tion  the  capsule  is  so  deeply  stained  that  the  bacillus  is  hidden;  by  careful 
treatment  with  a  weak  solution  of  acetic  acid  the  capsule  may  be  differen- 
tiated as  a  pale-red  or  violet  envelope  surrounding  the  deeply  stained  bacilli. 

Biotogical  Characters. — An  aer- 
obic and  facultative  anaerobic, 
non  liquefying,  non  motile  bacillus. 
Spore  formation  not  observed. 
Grows  in  the  usual  culture  media 
at  the  room  temperature.  The  cul- 
tures in  agar  or  upon  potato  are  very 
viscid  and  draw  out  into  long 
threads  when  touched  with  the  pla- 
tinum needle;  the  blood  of  an  ani- 
mal killed  by  inoculation  with  this 
bacillus  has  the  same  viscid  charac- 
ter. Upon  gelatin  plates  minute 
colonies  are  first  visible  at  the  end 
of  twenty  four  to  thirty-six  hours; 
later  the  deep  colonies  are  white, 
oval  masses  the  size  of  a  pin's  head ; 
the  superficial  colonies  attain  the 
size  of  a  lentil,  and  are  flattened, 
hemispherical  masses  with  a  porce- 
lain white  color.  In  gelatin  stick  Fia  143-~ Bacillus  capsulatus,  from  peritoneal 
Cultures  growth  occurs  to  the  bot-  exudate  of  an  inoculated  guinea-pig,  x  1,000. 
torn  of  the  line  of  puncture,  and  on  ^°*  a  photomicrograph.  (Ffeiffer.) 
the  surface  a  shining  white,  circular, 

arched  mass  forms  around  the  point  of  puncture,  resembling  the  growth  of 
Friedlander's  bacillus.  Upon  the  surface  of  agar,  at  37°  C  ,  at  the  end  of 
twenty-four  hours  a  thick,  soft  layer  of  a  pure  white  color  is  formed,  which 
is  very  viscid  and  resembles  the  growth  of  Micrococcus  tetragenus  upon  the 
same  medium.  Upon  potato  an  abundant  and  viscid,  shining,  yellowish- 
white  layer  is  quickly  developed. 

Pathogenesis. — Pathogenic  for  white  mice  and  for  house  mice,  which  die 
at  the  end  of  two  or  three  days  after  being  inoculated  at  the  root  of  the  tail 
with  a  small  quantity  of  a  pure  culture.  Inoculation  from  mouse  to  mouse 
increases  the  virulence  of  the  cultures.  At  the  autopsy  the  superficial  veins 
are  distended  with  blood,  the  inguinal  glands  enlarged,  the  spleen  consid- 
erably enlarged,  the  liver  and  kidneys  hyperaemic,  the  intestine  pale,  the 
heart  distended  with  blood,  which  usually  is  very  viscid  and  is  drawn  out 
into  threads  when  touched  with  the  platinum  needle.  The  bacilli  are  found 
in  the  blood  and  in  all  of  the  organs,  in  the  contents  of  the  peritoneum  and 
pleurae,  and  in  the  exudate  in  the  vicinity  of  the  point  of  inoculation. 
Pathogenic  also  for  guinea  pigs  and  for  pigeons;  guinea-pigs  are  infallibly 
killed  within  thirty-six  hours  by  the  injection  of  a  single  drop  of  a  bouillon 
culture,  twenty-four  hours  old,  into  the  cavity  of  the  abdomen;  the  blood 
contains  the  bacillus  in  enormous  numbers,  as  does  the  viscid  fluid  found  in 
the  peritoneal  cavity.  Rabbits  do  not  succumb  to  intraperitoneal  or  subcu- 
taneous inoculations,  but  are  killed  by  the  intravenous  injection  of  one 
cubic  centimetre  of  a  recent  bouillon  culture.  Putrefactive  changes  occur 
very  quickly  in  animals  killed  by  inoculation  with  this  bacillus. 

81.   BACILLUS  HYDROPHILTJS  FUSCUS. 

Obtained  by  Sanarelli  (1891)  from  the  lymph  of  frogs  suffering  from  a 
fatal  infectious  disease. 

Morphology. — Bacilli  with  rounded  ends,  usually  from  1  to  3  H  in  length; 
often  short  oval;  may  grow  out  into  filaments  of  12  to  20  jit  in  length. 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacillus.  Grows 
in  the  usual  culture  media  at  the  room  temperature.  In  gelatin  stick  cul- 


456 


BACILLI  WHICH  PRODUCE  SEPTICAEMIA 


<! 


FIG.  144.— Bacillus  hydroph'lus  fus- 
cus,  in  blood  of  triton.    (Sanarelli.) 


tures,  at  18°  to  20s  C.,  liquefaction  has  already  commenced  along-  the  line  of 
puncture  at  the  end  of  twelve  hours,  and  at  the  end  of  thirty-six  to  forty- 
eight  hours  half  of  the  gelatin  is  liquefied  in  funnel  shape;  on  the  third  or 
fourth  day  the  gelatin  is  completely  lique- 
fied, and  a  thick,  white,  flocculent  deposit 
is  seen  at  bottom  of  the  tube.  In  glycerin- 
agar,  at  37°  C.,  a  slight,  bluish,  diffuse 
fluorescence  is  seen  upon  the  surface  at  the 
end  of  twelve  hours,  and  soon  after  a  luxu- 
riant growth,  which  soon  covers  the  entire 
surface,  is  developed;  at  the  end  of  twenty- 
four  to  thirty-six  hours  large  gas  bubbles 
be,?in  to  form  in  the  agar;  gradually  the 
fluorescence  disappears,  the  surface  growth 
becomes  thicker  a/id  has  a  dirty-gray  color 
which  changes  later  to  brownish.  Blood 
serum  is  a  favorable  medium  and  is  rapidly 
liquefied  by  this  bacillus.  Upon  potato  the 
growth  is  most  characteristic.  At  the  end 
of  twelve  hours  a  thin,  straw-yellow  layer 
is  developed  along  the  impfstrich;  this 
gradually  becomes  vellow,  and  at  the  end 
of  four  to  five  days  has  a  brown  color,  resembling  that  of  the  glanders  bacil- 
lus upon  potato. 

Pathogenesis. — Pathogenic  for  frogs,  toads,  lizards,  and  oth  "cold- 
blooded" animals;  also  for  guinea-pigs,  rabbits,  dogs, 
cats,  mice,  chickens,  and  pigeons.  When  a  few  drops  of 
a  bouillon  culture  are  injected  into  the  muscles  of  the 
thigh,  swelling  and  redness  at  the  point  of  inoculation 
are  quickly  developed,  and  death  usually  occurs  in  eight 
to  ten  hours.  The  bacilli  are  found  in  great  numbers  in 
the  blood  and  in  all  of  the  organs.  Guinea  pigs  die  from 
general  infection  within  twelve  hours  after  receiving  a 
subcutaneous  injection  of  a  small  amount  of  a  pure  cul- 
ture; the  spleen  is  enlarged  and  the  liver  and  spleen  hy- 
peraemic;  an  extensive  inflammatory  oedema  in  the  vicin- 
ity of  the  inoculation  wound  is  frequently  observed;  the 
bacilli  are  very  numerous  in  the  blood  and  in  all  the  or- 
gans. Rabbits  die  in  five  to  six  hours  from  an  intravenous 
injection.  Adult  dogs  are  immune,  but  new-born  dogs 
(three  to  four  days  old)  die  infallibly,  after  receiving  a 
subcutaneous  injection  of  a  small  quantity  of  a  pure  cul- 
ture, in  twelve  to  thirty-six  hours.  Young  cats  also  suc- 
cumb to  similar  inoculations.  Chickens  and  pigeons  die 
within  five  to  seven  hours  after  receiving  an  intravenous 
injection,  but  resist  subcutaneous  injections. 

82.    BACILLUS  TENUIS  SPUTIGENUS. 

Obtained  by  Pansini  (1890)  from  sputum. 

Morphology.  Short  bacilli,  usually  in  pairs  and  sur- 
rounded by  a  capsule. 

Stains  by  Gram's  method. 

Biological  Characters.—  An  aerobic,  non-liquefying ', 
non  motile  bacillus.  Grows  in  nutrient  gelatin  at  the 
room  temperature.  Develops  abundantly  on  potato. 
Coagulates  milk  and  produces  an  acid  reaction  in  this 
medium. 

1  athogenesis.  -  Pathogenic  for  rabbits  and  white  rats;  not  for  guinea- 
pigs  or  for  white  mice  (in  small  doses). 


Fio.  145.-Bacillus 
hydrophilus  fuscus; 
culture  In  nutrieut 
gelatin,  end  of  tix- 
teen  hours.  (.Sana- 


IN  SUSCEPTIBLE  ANIMALS.  457 

83.    BACILLUS   OF   LASER. 

Obtained  by  Laser  (1892)  from  mice  which  succumbed  to  an  epidemic  dis- 
ease in  Frankel's  laboratory  at  Konigsberg. 

In  its  characters  this  bacillus  closely  resembles  the  bacillus  of  swine 
plague  (No.  65),  and  is  perhaps  identical  with  it. 

Morphology. — A  small  bacillus,  with  rounded  ends,  about  twice  as  long 
as  broad.  Has  flagella  both  at  the  extremities  and  sides. 

Stains  by  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  actively  motile  bacillus.  Spore  formation  not  observed.  Grows 
either  in  the  incubating  oven  or  at  the  room  temperature.  Thermal  death - 
point  65°  to  70°  C. — ten  minutes'  exposure.  Upon  gelatin  plates,  at  the  end 
of  two  days,  the  deep  colonies  are  spherical,  finely  granular,  and  brownish 
in  color;  the  superficial  are  transparent,  finely  granular,  and  leaf -like. 
In  gelatin  stick  cultures  growth  occurs  along  the  entire  line  of  puncture  as 
well  as  upon  the  surface.  At  the  end  of  three  days  a  considerable  evolution 
of  gas  is  usually  observed.  In  agar  an  abundant  development  is  seen  at  the 
end  of  twenty-four  hours  in  the  incubating  oven;  upon  the  surface  a  gray- 
ish-white, shining  layer  with  dentate  margins  is  formed  along  the  track  of 
the  needle.  In  bouillon,  at  37°  C.,  development  is  abundant  and  rapid;  a 
thin  film  is  formed  on  the  surface  at  the  end  of  the  second  day.  Upon  potato 
a  brownish  layer  is  formed  at  the  end  of  twenty-four  hours.  In  milk  an 
acid  reaction  is  produced. 

Pathogenesis.—  Pathogenic  for  field  mice,  guinea-pigs,  rabbits,  and 
pigeons.  The  bacillus  is  found  in  the  blood  and  various  organs  of  infected 
mice.  The  spleen  is  found  to  be  greatly  enlarged. 

84.   BACILLUS  TYPHI  MURIUM   (Loffler). 

Obtained  by  Loftier  (1889)  from  mice  which  died  in  his  laboratory  from 
an  epidemic  disease  due  to  this  bacillus. 

Morphology. — Short  bacilli,  resembling  the  bacillus  of  diphtheria  in 
pigeons,  and  varying  considerably  in  dimensions — like  the  bacillus  of 
typhoid  fever ;  grows  put  into  flexible  filaments. 

Stains  with  the  aniline  colors— best  with  Loffler's  solution  of  methylene 
blue. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Spore  formation  not  determined.  Has  flagella 
around  the  periphery  of  the  cells,  like  those  of  the  typhoid  bacillus,  and  ex- 
hibits similar  active  movements.  In  gelatin  stick  cultures,  at  the  room 
temperature,  growth  occurs  upon  the  surface,  at  the  end  of  forty-eight  hours, 
in  the  form  of  a  flat,  grayish-white,  round,  semi-transparent  mass  the  size  of 
a  pin's  head ;  later  the  surface  colony  increases  in  extent  and  has  more  or 
less  irregular  margins.  In  gelatin  plate  cultures  the  deep  colonies  are  at 
first  round,  slightly  granular,  transparent,  and  grayish ;  later  they  are  of  a 
yellowish-brown  color  and  decidedly  granular.  The  superficial  colonies  are 
very  granular  and  marked  by  delicate  lines — similar  to  colonies  of  the 
typhoid  bacillus.  Upon  agar  a  grayish- white  layer  is  developed  which  is 
not  at  all  characteristic.  Upon  potato  a  rather  thin,  whitish  layer  is  formed, 
and  around  this  the  potato  acquires  a  dirty  bluish-gray  color.  In  milk  an 
abundant  development  occurs,  and  a  decidedly  acid  reaction  is  produced 
without  causing  any  perceptible  change  in  the  appearance  of  the  fluid. 

Pathogenesis. — Pathogenic  for  white  mice,  which  die  in  from  one  to  two 
weeks  after  infection  ;  also  to  field  mice,  which  succumb  to  subcutaneous  in- 
jections of  a  pure  culture,  and  also,  in  from  eight  to  twelve  days,  when  fed 
upon  potato  cultures  or  bread  moistened  with  a  small  quantity  of  a  bouillon 
culture.  Loftier  believes  that  this  bacillus  may  be  used  for  the  destruction 
of  field  mice  in  grain  fields,  inasmuch  as  they  invariably  die  after  ingesting 
food  which  has  been  contaminated  with  it,  and  also  from  eating  the  bodies 
32 


4,58  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

of  other  mice  which  have  died  as  a  result  of  infection.  House  mice  are  also 
susceptible.  Rabbits,  guinea  pigs,  pigeons,  and  chickens  were  found  by 
Loftier  not  to  be  susceptible  to  infection  by  feeding. 

85.    BACILLUS   OP   CAZAL   AND   VAILLARD. 

Obtained  by  Cazal  and  Vaillard  (1891)  from  cheesy  nodules  upon  the 
peritoneum  and  in  the  pancreas  of  an  individual  who  died  in  the  hospital 
at  Val  de  Grace. 

Morphology. — Bacilli  with  rounded  ends,  but  little  longer  than  they  are 
broad ;  solitary,  in  pairs,  or  in  chains  of  ten  to  fifteen  or  more  elements. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method ;  the 
extremities  of  the  rods  are  more  deeply  stained  than,  the  central  portion — 
'*  polar  staining." 

Biological  characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacil  lus.  Does  not  form  spores.  Grows  in  the  usual  culture  media 
at  the  room  temperature — more  rapidly  in  the  incubating  oven  at  37°  C.  In 
gelatin  stick  cultures,  at  the  end  of  twenty-four  hours,  a  series  of  puncti- 
form,  white  colonies  is  developed  along  the  line  of  puncture ;  upon  the  sur- 
face development  is  more  abundant,  and  at  the  end  of  forty-eight  hours 
liquefaction  commences  ;  this  progresses  slowlv  from  above  downward, 
and  a  white,  flocculent  deposit  accumulates  at  tne  bottom  of  the  liquefied 
gelatin.  ^Upon  the  surface  of  agar,  at  the  end  of  twenty-four  hours  at  37° 
C.,  a  moist,  transparent,  opalescent  layer  is  developed,  which  rapidly  ex- 
tends over  the  entire  surface  ;  later  this  layer  becomes  somewhat  thicker, 
whitish,  and  cream-like  in  consistence,  without  losing  its  transparency. 
Upon  potato  a  thick,  prominent,  moist,  and  slightly  viscid  layer  is  devel- 
oped, which  at  first  has  a  pale-yellow  and  later  a  yellowish-brown  color. 
In  bouillon  development  is  abundant,  producing  a  milky  opacity  of  the 
liquid ;  a  thick,  flocculent  deposit  accumulates  at  the  bottom  of  the  tube  ; 
the  reaction  of  the  culture  liquid  becomes  very  alkaline.  All  of  the  cultures 
give  off  a  peculiar  odor,  slightly  ammoniacal  and  resembling  that  of  putrid 
urine.  The  cultures  retain  their  vitality  for  several  months— in  a  closed 
tube  for  more  than  a  year.  The  thermal  death-point  is  60°  C.  with  fifteen 
minutes'  exposure. 

Pathoqenesis.—  Pathogenic  for  rabbits  and  mice,  but  not  for  guinea-pigs. 
In  mice  death  occurs  from  general  infection,  at  the  end  of  forty  -eight  to 
sixty  hours,  from  the  subcutaneous  injection  of  one-eighth  cubic  centimetre 
of  a  recent  bouillon  culture.  In  rabbits  injection  of  one  cubic  centimetre 
into  the  circulation  causes  the  death  of  the  animal  in  thirty-six  to  fifty 
hours.  The  symptoms  induced  are  a  foetid  diarrhoea  and  paralysis  of  the 
extremities.  When  smaller  doses  are  injected  (0.5  cubic  centimetre)  a 
chronic  malady  is  developed,  characterized  at  the  outset  by  diarrhoea  and 
emaciation,  then  by  the  development  of  tumors  which  resemble  those  found 
in  the  man  from  whom  the  cultures  were  first  obtained.  These  tumors  are 
for  the  most  part  located  in  the  subcutaneous  connective  tissue ;  after  a  time 
they  attain  tne  size  of  a  chestnut  and  ulcerate,  allowing  the  escape  of  a 
K« -nil  thud,  purulent  material.  The  animals  usually  recover.  Similar  tumors 
are  developed  as  a  result  of  subcutaneous  injections  of  one  to  three  cubic 
centimetres  of  a  recent  bouillon  culture. 

86.  BACILLUS  OF  BABES  AND  OPRESCU. 

Obtained  by  Babes  and  Oprescu  (1891)  from  a  case  of  septicaemia  luumor- 
fficu  presenting  some  resemblance  to  exanthematic  typhus. 
Morphology.— In  agar  cultures  the  bacilli  are  from  0.4  to  0.5  /*  thick,  and 
are  frequently  united  in  pairs ;  associated  with  these  rod-shaped  bacteria  are 
orms  which  are  of  a  short  oval.    In  gelatin  cultures  oval  forms  are  more 
they  have  a  diameter  of  0.3  to  0.4  >u,  and  often  appear  to  be 
surrounded  by  a  capsule.     In  fresh  cultures  the  bacilli  are  often  in  form  of 


IN   SUSCEPTIBLE   ANIMALS.  459 

a  figure  8,  and  are  only  stained  at  the  point  of  contact  of  the  two  segments. 
In  potato  cultures  they  are  sometimes  elongated  and  swollen  at  one  ex- 
tremity. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, actively  motile  bacillus.  Spore  formation  not  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature — more  rapidly  at  37°  C.  In 
gelatin  stick  cultures  yellowish- white  colonies  are  developed  along  the  line 
of  puncture ;  at  the  bottom  these  may  have  a  diameter  of  one  to  two  millime- 
tres, and  they  have  a  brown  color.  Upon  the  surface  an  irregular,  lobulated, 
whitish,  translucent,  paraffin-like  layer  is  developed.  At  the  end  of  eight 
days  the  surface  growth  consists  of  large,  confluent,  transparent  plaques, 
with  irregular  outlines  and  crenated,  elevated  margins  ;  along  the  line  of 
puncture  large,  separate,  lenticular  or  spherical  colonies  are  seen  ;  these 
have  a  brownish-white  color.  At  the  end  of  two  months  the  surface  growth 
is  concentric  and  still  more  transparent,  while  the  colonies  near  the  surface 
have  become  almost  brown.  Upon  the  surface  of  agar,  at  37°  C.,  a  narrow 
band  is  developed  along  the  line  of  inoculation ;  above,  this  is  composed  of 
transparent,  shining,  flat,  round  colonies  having  a  diameter  of  one  milli- 
metre or  more ;  below,  the  colonies  are  confluent  and  form  a  transparent, 
whitish  layer.  In  glycerin-agar  development  is  still  more  abundant,  and 
may  already  be  perceived  at  the  end  of  twelve  hours.  Crystals  are  seen 
below  the  surface  in  agar  cultures  and  about  the  superficial  colonies  in  gela- 
tin. Upon  potato  a  uniform,  thin,  grayish,  verv  transparent  layer  is  de- 
veloped, which  sometimes  has  a  brownish-gray  tint.  At  the  end  of  a  few 
days  the  potato  acquires  a  brownish  color.  In  bouillon  cloudiness  of  the 
medium  is  apparent  at  the  end  of  ten  hours  ;  twenty-four  hours  later  a 
whitish  precipitate  is  seen  at  the  bottom  of  the  tube,  which  is  more  abun- 
dant when  the  culture  medium  contains  glucose;  later  a  thin  pellicle  is 
seen  upon  the  surface  and  the  bouillon  acquires  a  yellowish  color. 

Pathogenesis. — Recent  cultures  are  pathogenic  for  rabbits,  guinea-pigs, 
pigeons,  and  mice,  which  die  from  general  infection  in  from  two  to  four 
days.  Old  cultures  are  less  virulent. 

87.    BACILLUS  OF  LUCET. 

Obtained  by  Lucet  (1891)  from  chickens  and  turkeys  suffering  from  an 
infectious  form  of  septicaemia  characterized  by  dysenteric  discharges — "  Dy- 
senterie  epizootique  des  poules  et  des  dindes." 

Resembles  Bacillus  gallinarum  of  Klein,  and  is  perhaps  identical  with 
this  microorganism. 

Morphology.—  Short  bacilli,  from  1.2  to  1.8  /*  long,  usually  in  pairs. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, non-motilebaicillus.  Spore  formation  not  observed.  Grows  slowly  in 
the  usual  culture  media  at  the  room  temperature — more  rapidly  at  37°  C. 

In  gelatin  plates  small,  shining,  moist,  white,  circular  colonies  are  devel- 
oped, which  look  like  little  drops  of  wax ;  later  these  increase  in  size,  and 
especially  in  thickness,  forming  hemispherical  masses.  In  gelatin  stick  cul- 
tures grayish,  punctiform  colonies  are  developed  along  the  line  of  puncture, 
and  upon  the  surface  a  circular,  prominent,  whitish  plaque.  Streak  cultures 
upon  the  surface  of  gelatin  are  in  the  form  of  a  dirty-white  or  grayish- white, 
moist  streak,  with  regular  margins,  limited  to  the  line  of  inoculation,  but 
increasing  in  thickness  until  it  breaks  loose  and  slips  down  the  oblique  sur- 
face of  the  culture  medium.  The  deposit  which  collects  in  this  way  acquires, 
as  it  becomes  old,  in  the  deepest  portion  a  reddish  color.  Upon  agar  it  forms 
a  thick,  yellowish- white,  mucus-like  layer  with  straight  or  slightly  dentate 
margins.  In  bouillon  it  produces  a  decided  clouding  of  the  liquid,  and  an 
abundant  grayish,  pulverulent  sediment  accumulates  at  the  bottom  of  the 
tube ;  the  bouillon  after  a  time  becomes  transparent  above  this  sediment  and 


460  BACILLI  WHICH  PRODUCE   SEPTICAEMIA. 

is  viscid,  drawing  out  into  threads.  In  the  absence  of  oxygen  the  characters 
of  growth  are  the  same  as  in  its  presence.  The  cultures  acquire  an  alkaline 
reaction ;  they  are  sterilized  by  exposure  for  ten  minutes  to  a  temperature  of 
60°  C.  Does  not  grow  upon  potato. 

Pathogenesis. — Pathogenic  for  chickens  and  turkeys.  Not  pathogenic  for 
pigeons,  guinea-pigs,  or  rabbits  when  injected  subcutaneously  or  into  the 
peritoneal  cavity,  but  kills  rabbits  when  injected  into  a  vein.  In  the  in- 
fected fowls  the  bacilli  are  found  in  small  numbers  in  the  blood,  more  nu- 
merous in  the  kidneys  and  liver,  still  more  numerous  in  the  spleen,  and  in 
enormous  numbers  in  the  intestinal  mucus,  where  in  acute  cases  it  is  found 
almost  in  a  pure  culture.  Fowls  do  not  contract  the  disease  as  a  result  of 
the  ingestion  of  grains  soiled  with  cultures  of  the  bacillus,  but  become  in- 
fected when  fed  with  animal  food  to  which  a  pure  culture  has  been  added. 

88.   CAPSULE  BACILLUS  OF  LOEB. 

Obtained  from  a  case  of  keratomalacia  infantum  by  inoculating  culture 
media  with  a  little  of  the  softened  exudate  in  the  cornea. 

Morphology. — Resembles  Bacillus  capsulatus  of  Pfeiffer,  but  this  is  said 
to  be  somewhat  larger  and  thicker.  In  the  blood  of  mice,  however,  both 
bacilli  vary  considerably  in  size,  and  according  to  Loeb  it  was  not  possible 
to  determine  with  certainty  that  one  bacillus  was,  on  the  average,  larger 
than  the  other. 

In  staining  reactions,  also,  no  difference  was  observed — both  bacilli  stain 
with  the  usual  aniline  colors,  and  under  certain  circumstances  the  centre  of 
the  rods  is  less  deeply  stained  than  the  extremities. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, non-motile  bacillus.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  In  its  growth  in  culture  media  it  closely  resembles  Bacillus 
capsulatus  of  Pfeiffer  (No.  80). 

Pathogenesis. — Pathogenic  for  mice  and  for  guinea-pigs,  but  not  for  rab- 
bits and  pigeons ;  Pf  eiffer's  bacillus  is  pathogenic  for  these  animals. 


PLATE  VII. 

BACILLUS  OP  GLANDERS. 

IG.  1. — Bacillus  mallei  from  the  liver  of  a  field  mouse,  cover -glass  pre- 
paration. (Loffler.) 

FIG.  2.— Bacillus  mallei  from  a  recent  culture  upon  blood  serum-  (Lof 
fler.) 

FIG.  3. — Bacillus  mallei  in  section  of  spleen  of  a  field  mouse  dead  from 
glanders.  (Loffler.) 

FlG  4.— Culture  of  glanders  bacillus  upon  cooked  potato.     (Loftier.) 


r....,.,,  i.     :..i. 


VMKi?(Vs  IJACTKKIOLOCVY. 


Plate 


'% 


Fig.l. 


Fig.  2. 


X  " 


•       4       ^ 


H ACII.LITS  OF  GLAND EKS  (I.OKFFLER) 


XIII. 

PATHOGENIC  AEROBIC  BACILLI    NOT  DESCRIBED  IN 
PREVIOUS   SECTIONS. 

A  CONSIDERABLE  number  of  saprophytic  bacilli  are  pathogenic  for 
small  animals  when  injected  into  the  circulation,  or  subcutaneously, 
or  into  a  serous  cavity  in  considerable  quantity — one  to  five  cubic 
centimetres  or  more — but  fail  to  produce  any  appreciable  effect 
when  introduced  into  the  bodies  of  these  animals  in  minute  doses, 
and  do  not  multiply  in  the  blood  to  any  considerable  extent,  al- 
though in  fatal  cases  they  may  usually  be  recovered  in  cultures  from 
the  blood  and  tissues.  These  bacilli  are  pathogenic  by  reason  of  the 
toxic  ptomaines  produced  by  them,  or  because  of  local  inflammatory 
processes  which  they  induce,  or  for  both  of  these  reasons  combined. 
Some  of  them  may  also,  under  certain  circumstances,  multiply  in 
the  blood  and  thus  give  rise  to  septicaemia  as  well  as  to  toxaemia ; 
this  is  the  case,  for  example,  with  the  "  colon  bacillus  "  of  Escher- 
ich.  When  injected  in  considerable  quantity  into  the  circulation 
of  a  guinea-pig  it  causes  the  death  of  the  animal  within  twenty-four 
hours,  and  the  bacillus  is  found  in  the  blood  in  great  numbers  ;  but 
minute  amounts  injected  into  a  vein,  or  larger  amounts  injected 
subcutaneousiy,  do  not  usually  produce  general  infection.  It  is, 
therefore,  not  included  among  the  "bacilli  which  produce  septi- 
caemia in  susceptible  animals/'  There  is  reason  to  believe,  however, 
that  under  certain  circumstances  this  bacillus  may  have  sufficient 
pathogenic  potency  to  produce  a  genuine  septicaemia  in  guinea-pigs. 
Thus  the  original  cultures  of  Brieger's  bacillus,  which  appears  to  be 
a  variety  or  the  colon  bacillus,  are  reported  to  have  produced  fatal 
septicaemia  in  guinea-pigs  when  injected  subcutaneousiy  in  small 
amounts.  A  strict  division  into  pathogenic  bacilli  which  produce 
general  blood  infection — septicaemia — and  those  which  produce  a 
fatal  result  owing  to  the  production  of  toxic  chemical  substances  is 
not  possible;  for  many  pathogenic  bacteria  produce  general  infection 
when  injected  in  comparatively  large  doses,  and  at  the  same  time 
give  rise  to  symptoms  of  toxaemia  ;  or  general  infection  may  occur 
in  animals  of  one  species,  and  fatal  toxaemia  without  septicaemia  in 


462  PATHOGENIC   AEROBIC  BACILLI 

those  of  another  species.  Many  of  the  bacilli  described  in  the  pre- 
sent section  are  common  saprophytes,  which  have  been  shown  by 
laboratory  experiments  to  be  pathogenic  for  certain  animals  when 
introduced  into  their  bodies  in  a  certain  amount,  which  differs  greatly 
for  different  bacteria  and  for  different  species  of  animals.  The  ex- 
periments of  Cheyne  and  others  show  how  largely  the  pathogenic 
power  of  saprophytic  bacteria  depends  upon  the  quantity  of  a  cul- 
ture which  is  injected,  as  well  as  upon  the  age  of  the  culture  and 
the  seat  of  the  inoculation — in  the  blood,  the  abdominal  cavity,  the 
subcutaneous  tissues,  or  the  muscles.  And  the  bacteriologist  named 
has  also  shown  that  pathogenic  power  depends,  in  some  instances  at 
least,  upon  the  combined  action  of  the  toxic  substances  introduced 
in  the  first  instance  and  of  the  living  bacteria.  Thus  Cheyne  found 
that  one-tenth  of  a  cubic  centimetre  of  a  bouillon  culture  of  Proteus 
vulgaris  injected  into  the  dorsal  muscles  of  a  rabbit  infallibly  caused 
its  death  within  forty-eight  hours,  but  when  the  dose  was  reduced 
to  one-fortieth  cubic  centimetre  the  animal  recovered.  But  if  to 
this  amount  (one-fortieth  cubic  centimetre)  he  added  one  cubic  cen- 
timetre of  a  sterilized  (by  heat)  culture  of  the  same  bacillus  instead 
of  diluting  with  distilled  water,  and  injected  the  mixture  into  the 
dorsal  muscles  of  a  rabbit,  death  occurred  in  every  experiment 
within  forty-eight  hours.  The  sterilized  culture  injected  by  itself 
produced  no  effect  in  this  dose  (one  cubic  centimetre),  and  Cheyne 
believes  that  the  fatal  result  in  these  experiments  was  due  to  the 
fact  that  the  toxic  products  present  in  the  sterilized  culture  over- 
came the  natural  resisting  powers  of  the  tissues  and  enabled  the 
bacillus  to  multiply  over  a  larger  area  than  would  otherwise  have 
been  the  case.  As  a  result  of  this,  toxic  substances  were  produced  in 
the  body  of  the  animal  in  sufficient  quantity  to  cause  general  toxae- 
mia and  death  ;  whereas  the  bacilli  alone,  in  the  dose  mentioned, 
were  not  able  to  invade  the  tissues  in  the  vicinity  of  the  point  of 
inoculation,  and  gave  rise  to  a  local  abscess  only.  The  same  ex- 
planation is  probably  true  for  very  many  of  the  saprophytic  bacteria 
which  have  been  shown  to  possess  pathogenic  power  ;  and  it  is  prob- 
able that  many  of  those  which  are  now  classed  by  bacteriologists  as 
non-pathogenic  would  prove  to  be  pathogenic  in  the  same  way  if 
thoroughly  tested  upon  various  species  of  animals,  although  it  might 
be  necessary  to  use  unusually  large  doses  to  accomplish  the  same 
result. 

89.    BACILLUS   COLI  COMMUNIS. 

Synonyms. — Bacterium  coli  commune  (Escherich) ;  Colon  bacillus 
of  Escherich  ;  Emmerich's  bacillus  (Bacillus  Neapolitanus).  Prob* 
ably  identical  with  Bacillus  cavicida  (Brieger's  bacillus). 


NOT  DESCRIBED  IN  PREVIOUS  SECTIONS.  463 

Obtained  by  Emmerich  (1885)  from  the  blood,  various  organs,  and 
the  alvine  discharges  of  cholera  patients  at  Naples ;  by  Weisser 
(1886)  from  normal  and  abnormal  human  faeces,  from  the  air,  and 
from  putrefying  infusions ;  by  Escherich  (1886)  from  the  fasces  of 
healthy  children  ;  since  shown  to  be  commonly  present  in  the  alvine 
discharges  of  healthy  men,  and  probably  of  many  of  the  lower  ani- 
mals. Found  by  the  writer'in  the  blood  and  various  organs  of  yellow- 
fever  cadavers,  in  Havana  (1888  and  1889). 

Numerous  varieties  have  been  cultivated  by  different  bacteriolo- 
gists, which  vary  in  pathogenic  power  and  to  some  extent  in  their 
growth  in  various  culture  media ;  but  the  differences  described  are 
not  sufficiently  characteristic  or  constant  to  justify  us  in  considering 
them  as  distinct  species. 

Morphology. — Differs  considerably  in  its  morphology  as  obtained 
from  different  sources  and  in  various  culture  media.     The  typical 
form  is  that  of  short  rods  with  rounded  ends,  from  two  to  three  /*  in 
length  and  0.4  to  0.6  yu  broad ;  but  under  certain  cir- 
cumstances the  length  does  not  exceed  the  breadth — 
about  0. 5  /* — and  it  might  be  mistaken  for  a  micrococ- 
cus  ;  again  the  prevailing  form  in  a  culture  is  a  short 
oval ;  filaments  of  five  /*  or  more  in  length  are  often 
observed  in  cultures,  associated  with  short  rods  or  oval      FIG.  146.— Ba- 
cells.     The  bacilli  are  frequently  united  in  pairs.     The   £"™&  co"  j^J; 
presence  of  spores  has  not  been  demonstrated.     In  un-   (Escherich.) 
favorable  culture  media  the  bacilli,  in  stained  prepara- 
tions, may  present  unstained  places,  which  are  supposed  by  Escherich 
to  be  due  to  degenerative  changes  in  the  protoplasm.    Under  certain 
circumstances  some  of  the  rods  in  a  pure  culture  have  been  observed 
by  Escherich  to  present  spherical,  unstained  portions  at  one  or  both 
extremities,  which  closely  resemble  spores, but  which  he  was  not  able 
to  stain  by  the  methods  usually  employed  for  staining  spores,  and 
which  he  is  inclined  to  regard  as  "  involution  forms." 

This  bacillus  stains  readily  with  the  aniline  colors  usually  em- 
ployed by  bacteriologists,  but  quickly  parts  with  its  color  when 
treated  with  iodine  solution — Gram's  method — or  with  diluted  al- 
cohol. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic, 
non-liquefying  bacillus.  Sometimes  exhibits  independent  move- 
ments, which  are  not  very  active.  One  rod  of  a  pair,  in  a  hanging- 
drop  culture,  may  advance  slowly  with  a  to-and-fro  movement, 
while  the  other  follows  as  if  attached  to  it  by  an  invisible  band 
(Escherich).  The  writer's  personal  observations  lead  him  to  believe 
that,  as  a  rule,  this  bacillus  does  not  exhibit  independent  movements. 
Does  not  form  spores.  Grows  in  various  culture  media  at  the  room 


464  PATHOGENIC  AEROBIC  BACILLI 

temperature — more  rapidly  in  the  incubating  oven.     Grows  in  a  de- 
cidedly acid  medium. 

In  gelatin  plates  colonies  are  developed  in  from  twenty-four  to 
forty-eight  hours,  which  va,ry  considerably  in  their  appearance  ac- 
cording to  their  age,  and  in  different  cultures  in  the  same  medium. 
The  deep  colonies  are  usually  spherical  and  at  first  are  transparent, 
homogeneous,  and  of  a  pale-straw  or  amber  color  by  transmitted 
light ;  later  they  frequently  have  a  dark-brown,  opaque  central  por- 
tion surrounded  by  a  more  transparent  peripheral  zone  ;  or  they  may 
be  coarsely  granular  and  opaque ;  sometimes  they  have  a  long-oval 
or  "whetstone"  form.  The  superficial  colonies  differ  still  more  in 
appearance  ;  very  young  colonies  by  transmitted  light  often  resemble 
little  drops  of  water  or  fragments  of  broken  glass  ;  when  they  have 
sufficient  space  for  their  development  they  quickly  increase  in  size, 
and  may  attain  a  diameter  of  three  to  four  centimetres  ;  the  central 
portion  is  thickest,  and  is  often  marked  by  a  spherical  nucleus  of  a 
dark-brown  color  when  the  colony  has  started  below  the  surface  of 
the  gelatin  ;  the  margins  are  thin  and  transparent,  the  thickness 
gradually  increasing  towards  the  centre,  as  does  also  the  color,  which 
by  transmitted  light  varies  from  light  straw  color  or  amber  to  a  dark 
brown.  The  outlines  of  superficial  colonies  are  more  or  less  irregular, 
and  the  surface  may  be  marked  by  ridges,  fissures,  or  concentric 
rings,  or  may  be  granular.  The  writer  has  observed  colonies  re- 
sembling a  rosette,  or  a  daisy  with  expanded  petals.  Escherich 
speaks  of  colonies  which  present  star-shaped  figures  surrounded  by 
concentric  rings. 

In  gelatin  stick  cultures  the  growth  upon  the  surface  is  rather 
dry,  and  may  be  quite  thin,  extending  over  the  entire  surface  of  the 
gelatin,  or  it  may  be  thicker  with  irregular,  leaf -like  outlines  and 
with  superficial  incrustations  or  concentric  annular  markings.  An 
abundant  development  occurs  all  along  the  line  of  puncture,  which 
in  the  deeper  portion  of  the  gelatin  is  made  up  of  more  or  less  closely 
crowded  colonies  ;  these  are  white  by  reflected  light,  and  of  an  am- 
ber or  light-brown  color  by  transmitted  light ;  later  they  may  become 
granular  and  opaque.  Frequently  a  diffused  cloudy  appearance  is 
observed  near  the  surface  of  the  gelatin,  and  under  certain  circum- 
stances branching,  moss-like  tufts  develop  at  intervals  along  the  line 
of  growth.  One  or  more  gas  bubbles  may  often  be  seen  in  recent 
stick  cultures  in  gelatin. 

Upon  nutrient  agar  and  blood  serum,  in  the  incubating  oven,  an 
abundant,  soft,  white  layer  is  quickly  developed.  Upon  potato  an 
abundant,  soft,  shining  layer  of  a  brownish-yellow  color  is  developed. 
The  growth  upon  potato  differs  considerably,  according  to  the  age  of 
the  potato.  According  to  Escherich,  upon  old  potatoes  there  may 


NOT  DESCRIBED   IN   PREVIOUS   SECTIONS. 


465 


be  no  growth,  or  it  may  be  scanty  and  of  a  white  color.  In  milk,  at 
37°  C.,  an  acid  reaction  and  coagulation  of  the  casein  are  produced  at 
the  end  of  eight  or  ten  days.  In  the  absence  of  oxygen  this  bacillus 
is  able  to  grow  in  solutions  containing  grape  sugar  (Escherich).  In 
bouillon  it  grows  rapidly,  producing  a  milky  opacity  of  the  culture 
liquid.  The  thermal  death-point  of  Emmerich/s  bacillus,  and  of  the 
colon  bacillus  from  faeces,  was  found  by  Weisser  to  be  60°  C.,  the 
time  of  exposure  being  ten  minutes.  The  writer  has  obtained  corre- 
sponding results.  Weisser  found  that  when  the  bacilli  from  a  bouil- 
lon culture  were  dried  upon  thin  glass  covers  they  failed  to  grow 


FIG.  147.  F;o.  148. 

FIG.  147.— Bacillus  coli  communis  in  nutrient  gelatin  containing  twenty  percent  of  gelatin,  end 
of  two  week*,  showing  moss-like  tufts  along  the  line  of  growth.  (Sternberg.) 

FIG.  148.— A  portion  of  the  growth  shown  in  Fig  147,  at  a,  magnified  about  sir  diameters. 
From  a  photograph.  (Sternberg.) 

after  twenty-four  hours.  These  results  give  confirmation  to  the 
view  that  the  bacillus  under  consideration  does  not  form  spores. 
This  view  receives  further  support  from  the  experiments  of  Wal- 
liczek  (1894),  who  found  that  when  dried  upon  pieces  of  sterile  filter 
paper  the  bacillus  failed  to  grow  at  the  end  of  eighteen  hours. 

Pathogenesis. — Comparatively  small  amounts  of  a  pure  culture 
of  the  colon  bacillus  injected  into  the  circulation  of  a  guinea-pig 
usually  cause  the  death  of  the  animal  in  from  one  to  three  days,  and 
the  bacillus  is  found  in  considerable  numbers  in  its  blood.  But  when 


4GG  PATHOGENIC   AEROBIC   BACILLI 

injected  subcutaneously  or  into  the  peritoneal  cavity  of  rabbits  or 
guinea-pigs,  a  fatal  termination  depends  largely  on  the  quantity  in- 
jected ;  and  although  the  bacillus  may  be  obtained  in  cultures  from 
the  blood  and  the  parenchyma  of  the  various  organs,  it  is  not  present 
in  large  numbers,  and  death  appears  to  be  due  to  toxaemia  rather  than 
to  septicaemia.  Mice  are  not  susceptible  to  infection  by  subcutaneous 
injections.  Small  quantities  injected  beneath  the  skin  of  guinea-pigs 
usually  produce  a  local  abscess  only  ;  larger  amounts — two  to  five 
cubic  centimetres — frequently  produce  a  fatal  result,  with  symptoms 
and  pathological  appearances  corresponding  with  those  resulting 
from  intravenous  injection.  These  are  fever,  developed  soon  after 
the  injection,  diarrhoea,  and  symptoms  of  collapse  appearing  shortly 
before  death.  At  the  autopsy  the  liver  and  spleen  appear  normal,  or 
nearly  so ;  the  kidneys  are  congested  and  may  present  scattered 
punctiform  ecchymoses  (Weisser).  According  to  Escherich,  the 
spleen  is  often  somewhat  enlarged.  The  small  intestine  is  hyper- 
semic,  especially  in  its  upper  portion,  and  the  peritoneal  layer  pre- 
sents a  rosy  color ;  the  mucous  membrane  gives  evidence  of  more 
or  less  intense  catarrhal  inflammation,  and  contains  mucus,  often 
slightly  mixed  with  blood.  In  rabbits  death  occurs  at  a  somewhat 
later  date,  and  diarrhoea  is  a  common  symptom.  In  dogs  the  subcu- 
taneous injection  of  a  considerable  quantity  of  a  pure  culture  may 
give  rise  to  an  extensive  local  abscess. 

In  human  pathology  the  colon  bacillus  plays  an  important  role. 
It  is  concerned  in  the  etiology  of  a  considerable  proportion  of  the 
cases  of  cystitis  and  of  pyelonephritis,  and  peritonitis  resulting  from 
perforation.  It  appears  to  be  the  cause  of  certain  affections  of  the 
anal  region  (Hartmann  and  Lieffring).  It  has  been  obtained  in  pure 
culture  from  abscesses  in  various  parts  of  the  body,  from  the  valves 
of  the  heart  in  endocarditis,  from  the  pleural  cavity  in  empyema,  etc. 
It  has  also  been  found  in  the  blood,  as  a  result  of  general  infection 
following  cystitis  and  pyelonephritis  (Sittmann  and  Barnow). 

Varieties. — Booker,  in  his  extended  studies  relating  to  the  bac- 
teria present  in  the  faeces  of  infants  suffering  from  summer  diarrhoea, 
has  isolated  seven  varieties  "  which  closely  resemble  Bacterium  coli 
commune  in  morphology  and  growth  in  agar,  neutral  gelatin,  and 
potato,  but  by  means  of  other  tests  a  distinction  can  be  made  between 
them." 

Some  of  the  pathogenic  bacteria  heretofore  described  are  also 
closely  allied  to  the  "  colon  bacillus  "  and  by  some  bacteriologists  are 
supposed  to  belong  to  the  same  group—  i.e.,  to  be  varieties  of  the 
same  species  rather  than  independent  species  with  fixed  characters. 
Whatever  may  t>e  the  remote  relationship,  the  typhoid  group,  the  hog- 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  467 

cholera  group,  the  Bacillus  typhi  murium  of  Loffler,  the  bacillus  of 
Laser,  the  Bacillus  euteritidis  of  Gartner,  and  other  similar  bacilli 
appear  to  be  differentiated  from  one  another  by  characters  which 
justify  their  description  under  separate  names.  Still  it  is  difficult  to 
fix  upon  any  one  of  these  characters  to  which  specific  value  can  be 
attached ;  and,  in  view  of  the  many  varieties  found  in  nature  or  pro- 
duced artificially  in  laboratory  experiments,  we  are  not  justified  in 
asserting  that  our  classification  of  these  low  organisms  has  any  sub- 
stantial scientific  foundation.  The  difficulties  attending  an  attempt 
to  establish  specific  characters  are  well  illustrated  by  the  extensive 
literature  relating  to  the  differentiation  of  bacilli  belonging  to  the 
typhoid  group  from  those  belonging  to  the  colon  group.  The  main 
points  upon  which  the  distinction  must  depend  have  been  referred  to 
in  the  section  devoted  to  the  typhoid  bacillus. 

Fremlin  (1893)  has  made  a  comparative  study  of  the  colon  bacil- 
lus from  various  sources.  He  finds  the  common  characters  of  gas 
production  in  media  containing  sugar  and  coagulation  of  milk.  Cul- 
tivated from  different  animals  the  morphology  is  the  same,  but  there 
are  differences  as  regards  motility.  The  most  active  movements  are 
said  to  be  exhibited  in  the  bacillus  from  man,  while  the  variety  ob- 
tained from  the  intestines  of  rabbits  showed  scarcely  any  movements. 
The  different  varieties  displayed  considerable  differences  in  their 
growth  upon  potato. 

Dreyfuss  (1894)  finds  decided  differences  in  the  pathogenic  viru- 
lence of  the  colon  bacillus  from  healthy  individuals  and  from  those 
suffering  from  various  intestinal  disorders.  A  culture  from  the  dis- 
charges of  a  fatal  case  of  cholera  nostras  proved  to  be  exceptionally 
virulent — tested  by  intraperitoneal  injections  in  guinea-pigs.  Gilbert 
(1895),  as  a  result  of  his  extended  researches,  concludes  that  there  are 
five  principal  types  among  the  bacilli  most  nearly  related  to  the  colon 
bacillus:  1st.  Bacilli  which  differ  from  the  colon  bacillus  by  their 
being  non-motile.  This  type  includes  two  varieties :  one  gives  thick 
yellowish  colonies  upon  gelatin  plates  and  numerous  gas  bubbles  on 
potato — this  is  the  bacille  lactique  of  Pasteur  and  the  Bacillus  lactis 
aerogenes  of  Escherich ;  the  other  gives  thin,  bluish-white  colonies 
and  includes  the  bacille  de  rendocardite  of  Gilbert  and  Lion.  3d. 
Bacilli  which  differ  from  the  colon  bacillus  by  the  fact  that  cultures 
do  not  give  the  indol  reaction.  3d.  Bacilli  which  do  not  cause  the 
fermentation  of  lactose.  4th.  Bacilli  which  are  not  motile  and 
do  not  ferment  lactose.  5th.  Bacilli  which  are  not  motile,  do  not 
give  the  indol  reaction,  and  do  not  ferment  lactose. 

Theobald  Smith  (1895)  gives  the  following  account  of  his  method 
of  detecting  bacilli  of  the  "colon  group  "  in  water  : 


468  PATHOGENIC  AEROBIC   BACILLI 

4  *  The  method  followed  by  the  writer  in  the  general  bacteriological  exam- 
ination of  water  consists,  first,  in  the  preparation  of  gelatin  plates  for  the 
usual  enumeration  ;  and,  second,  in  the  addition  to  every  one  of  ten  fermen- 
tation tubes,  containing  a  one-per-cent  dextrose  bouillon,  a  certain  quantity 
of  water.  This  is  added  most  easily  by  first  diluting  the  water,  so  that  one  or 
two  cubic  centimetres  are  equivalent  to  the  quantity  which  it  is  desired  to  add 
to  each  tube.  Pipettes  graduated  by  drops  are  convenient,  but  not  so  accurate. 
In  case  of  ground  water  it  is  well  to  prepare  in  addition  a  flask  containing 
fifty  to  one  hundred  cubic  centimetres  of  the  water,  and  an  equal,  or  greater, 
quantity  of  bouillon,  to  which  sugar  is  not  added.  Plates  may  be  prepared 
from  this  flask  after  sixteen  to  twenty -four  hours.  When  gas  begins  to  ap- 
pear in  the  fermentation  tubes,  the  amount  accumulated  at  the  end  of  each 
twenty-four  hours  should  be  marked  with  a  glass  pencil  on  the  tube.  From 
these  tubes,  which  contain  fifty  to  sixty  per  cent  of  gas  on  the  third  day,  and 
are  very  strongly  acid,  plates  may  be  prepared  to  confirm  the  indications  of 
Bacillus  coli.  This,  however,  is  not  essential,  for  the  writer  has  found  as 
yet  no  species  having  these  fermentative  characters  which  is  not  one  of  the 
following  :  Bacillus  coli,  Bacillus  lactis  aerogenes,  Bacillus  enteriditis,  Bacil- 
lus typhi  murium,  Bacillus  cholera?  suis.  The  three  last-mentioned  species 
are  probably  as  rare  in  water  as  Bacillus  typhosus  itself. 

* '  My  own  experience  coincides  with  that  of  Matthews  when  he  states  that 
ninety -two  per  cent  of  all  bacteria  in  ground  water  are  suppressed  in  the 
thermostat.  While  the  addition  of  0.5  cubic  centimetre,  or  even  more,  of 
such  water  may  fail  to  produce  cloudiness  in  any  of  the  series  of  fermenta- 
tion tubes,  the  same  quantity,  or  less,  of  surface  water  never  fails  to  infect 
the  tubes." 

BACILLUS   d  OF   BOOKER. 

"  Found  in  two  cases  of  cholera  infantum  and  the  predominating  form  in 
one  serious  case  of  catarrhal  enteritis. 

14  Morphology.— Resembles  Bacterium  coli  commune. 

**  Groivth  in  Colonies. — Gelatin :  Colonies  grow  luxuriantly  in  gelatin,and 
thrive  in  acid  and  sugar  gelatin  equally  as  well  as  in  neutral  gelatin.  In 
the  latter  the  colonies  closely  resemble,  but  are  not  identical  with,  the  Bac- 
terium coli  commune.  In  acid  gelatin  they  ditt'er  very  much  from  Bacterium 
coli  commune.  The  colonies  spread  extensively  and  are  bluish-white  with 
concentric  rings.  Slightly  magnified,  they  have  a  large,  uniform,  yellow 
central  zone  surrounded  by  a  Border  composed  of  perpendicular  threads 
placed  thickly  together.  Sometimes  a  series  of  these  rings  appear  with  inter- 
vening yellow  rings.- 

"Agar:  The  colonies  are  round,  spread  out,  and  blue  or  bluish- white. 
Slightly  magnified,  they  have  a  pale-yellow  color. 

'  Stab  Cultures — Gelatin:  In  sugar  gelatin  the  surface  growth  has  a 
nearly  colorless  centre  surrounded  by  a  thick  border  with  an  outer  edge  of 
fine,  hair-like  fringe ;  the  growth  along  the  line  of  inoculation  is  fine  and  deli- 
cate. In  neutral  gelatin  the  growth  is  not  so  luxuriant  as  on  sugar  gelatin ; 
on  the  surface  it  is  thick  and  white,  with  a  delicate  stalk  in  the  depth. 

"Agar:  Thick  white  surface  growth  with  a  well-developed  stalk  in  the 
depth. 

"Potato:  Luxuriant  yellow,  glistening,  moist,  and  slightly  raised  sur- 
face, with  well-defined  Orders. 

11  Action  on  Milk. — Coagulated  into  a  gelatinous  coagulum  in  twenty-four 
hours  at  88*  G.,  and  into  a  solid  clot  in  two  days. 

"  Milk  Litmus  Reaction. — Milk  colored  blue  with  litmus  is  changed  to 
light  pink  in  twenty-four  hours  at  38°  C.  The  pink  color  gradually  fades, 
and  by  the  second  or  third  day  is  white  or  cream  color  with  a  thin  layer  of 
pint  on  top.  The  pink  color  extends  in  a  few  days  about  one-half  down  the 
clot. 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  469 

Temperature.— Grows  best  about  38°  C. 
"  Spores  have  not  been  observed. 

"  Gas  Production. — Gas  bubbles  are  produced  in  milk;  not  observed  on 
potato." 

BACILLUS   6   OF   BOOKER. 

* '  Found  as  the  predominating  form  in  two  cases  of  dysentery  one  of 
which  was  fatal  and  the  other  a  mild  case. 

"  Morphology. — Resembles  Bacterium  coli  commune. 

"  Growth  in  Colonies. — Gelatin  :  The  colony  growth  varies  considerably 
with  slight  difference  in  the  gelatin.  In  ten-per-cent  neural  gelatin  the  colo- 
nies resemble  those  of  Bacterium  coli  commune.  On  the  second  or  third 
day,  when  the  colonies  have  just  broken  through  the  surface  and  are  spread 
out,  it  is  impossible  to  distinguish  one  variety  from  the  other,  but  as  the 
colonies  grow  older  a  difference  can  generally  be  recognized.  In  sugar  and 
acid  gelatin  the  colonies  have  a  clear  centre  with  white  border ;  slightly 
magnified,  a  uniform  brown  centre  surrounded  by  a  brown  zone  composed 
of  fine,  needle- like  rays  perpendicular  to  the  border.  After  cultivating  for  a 
few  generations  on  acid  and  sugar  gelatin  the  colonies  cease  to  develop,  and 
either  grow  in  very  small  colonies  or  do  not  grow  at  all.  The  activity  is  re- 
gained if  cultivated  on  neutral  gelatin. 

"  Agar :  Colonies  are  large,  round,  and  have  a  mother-of-pearl  appearance. 
Slightly  magnified,  a  uniform  yellow  color. 

"  Stab  Cultures. — Agar:  Luxuriant,  nearly  colorless  surface  growth,  with 
well-developed  stalk  along  the  line  of  inoculation  in  the  depth. 

"Potato:  Golden-yellow,  glistening,  slightly  raised  surface  with  well-de- 
fined borders. 

' '  Action  on  Milk.— Milk  becomes  gelatinous  in  twenty-four  hours  at  38°  C. , 
and  in  a  few  days  a  solid  coagulum  is  formed.  Milk  colored  blue  with  lit- 
mus is  reduced  to  white  or  cream  color  in  twenty-four  to  forty-eight  hours 
at  38°  C.,  with  a  thin  layer  of  pink  at  the  top  of  the  culture.  The  pink  color 
gradually  extends  lower  in  the  coagulum. 

"  Temperature.—  Thrives  best  at  about  38°  C. 

"  Spores  have  not  been  observed. 

"  Gas  Production.— Occurs  in  milk,  but  not  seen  in  potato  cultures. 

"  Relation  to  Gelatin. — Does  not  liquefy  gelatin. 

"  Resemblance. — Resembles  Bacterium  coli  commune  and  bacillus  d;  dif- 
fering from  the  former  in  the  character  of  the  colony  growth  on  acid  and 
sugar  gelatin,  and  in  ceasing  to  develop  in  these  media  after  several  genera- 
tions. It  differs  from  bacillus  d  in  this  latter  respect." 

BACILLUS  /  OF  BOOKER. 

"  Found  in  one  case  of  cholera  infantum  and  one  case  of  catarrhal  ente- 
ritis. 

"  Morphology. — Resembles  Bacterium  coli  commune. 

"  Growth  in  Colonies.—  Gelatin:  It  is  difficult  to  distinguish  the  colony 
growth  from  the  Bacterium  coli  commune.  There  is  often  a  difference  in  the 
colonies  planted  at  the  same  time  and  kept  under  similar  conditions,  but  it 
is  not  very  marked  nor  always  the  same  kind  of  difference.  The  tendency 
to  concentric  rings  is  greater  in  this  variety.  The  colonies  develop  some- 
what better  on  neutral  and  sugar  gelatin  than  on  acid  gelatin. 

' '  Agar :  The  colonies  are  large,  round,  and  bluish- white.  Slightly  magni- 
fied, a  light-yellow  color. 

4 '  Stab  Cultures.  —Gelatin :  The  culture  is  spread  over  the  surface  and  has 
a  mist-like  appearance ;  in  the  depth  along  the  line  of  inoculation  is  a  deli- 
cate stalk.  , 

"Agar:  Thick,  luxuriant,  white  surface  growth,  with  a  well-developed 
stalk  along  the  line  of  inoculation  in  the  depth. 


470  PATHOGENIC   AEROBIC   BACILLI 

"  Potato  :  Bright-yellow,  glistening,  moist  surface  with  well-defined  bor- 
ders, and  but  slightly  raised  above  the  siirrounding  potato. 

"  Action  on  Milk  and  Litmus  Reaction. — Milk  is  coagulated  into  a  solid 
clot  in  twenty-four  hours  at  38°  C.  Milk  colored  blue  with  litmus  is  changed 
to  pink  in  twenty-four  hours  at  38°  C. ,  and  in  forty-eight  hours  is  reduced  to 
white  or  cream  color  with  a  thin  pink  layer  on  top. 

"  Gas  Production. — Gas  bubbles  arise  in  milk  cultures,  but  they  have  not 
been  observed  on  potato  cultures. 

"  Temperature.— Grows  better  at  38°  C. 

'*  Spores  have  not  been  observed. 

"Relation  to  Gelatin.— Does  not  liquefy  gelatin. 

"Resemblance. — It  closely  resembles  Bacterium  coli  commune  and  Brie- 
ger's bacillus  in  the  character  of  its  growth  upon  different  media,  but  is  readily- 
distinguished  from  both,  as  is  also  Brieger's  bacillus  from  the  Bacterium  coli 
commune,  by  the  following  differential  test  recently  made  known  by  Dr. 
Mall.  Yellow  elastic  tissue  from  the  ligamentum  nuchae  of  an  ox  is  cut  into 
fine  bits  and  placed  in  test  tubes  containing  water  with  ten-per-cent  bouillon 
and  one-per-cent  sugar,  and  sterilized  from  one  and  one-half  to  two 
hours  at  a  time  for  three  consecutive  days.  Into  this  is  inoculated  two 
species  of  bacteria,  one  of  which  is  the  bacterium  under  observation, 
the  other  a  bacillus  found  in  garden  earth.  The  latter  bacillus  is  anaerobic, 
grows  in  hydrogen,  nitrogen,  and  ordinary  illuminating  gas,  in  the  bottom 
of  bouillon,  in  the  depth  but  not  on  the  surface  of  agar  stab  cultures,  and 
not  at  all  in  gelatin  stab  cultures.  It  has  a  spore  in  one  end  making  a  knob 
bacillus.  Different  species  of  bacteria — Streptococcus  Indicus,  tetragenus, 
cholera,  swine  plague,  Bacterium  lactis  aerogenes,  Bacterium  coli  commune, 
Brieger's  bacillus,  and  a  number  of  varieties  of  bacteria  which  I  have  iso- 
lated from  the  faeces — were  inoculated  with  the  head  bacillus  into  the  above- 
described  elastic- tissue  tubes.  The  tubes  inoculated  with  Brieger's  bacillus 
developed  a  beautiful  purple  tint,  which  started  as  a  narrow  ring  at  the  top 
of  the  culture,  gradually  extending  downward  and  deepening  in  color  until 
the  whole  tube  had  a  dark-purple  color.  This  color  reaction  began  in  five  to 
fourteen  days,  and  was  constantly  present  in  a  large  number  of  tests.  Tubes 
inoculated  with  bacillus  /  gave  a  much  fainter  purple  color,  which  was 
longer  in  appearing  and  never  became  so  dark  as  with  Brieger's  bacillus. 

"Tubes  inoculated  with  the  other  species  of  bacteria  above  mentioned  gave 
no  color  change  and  remained  similar  to  control.  Bacillus  /  also  shows  a 
slight  difference  from  Bacterium  coli  commune  in  coagulating  milk  and  re- 
ducing litmus  more  rapidly,  and  appears  to  produce  moreactive  fermentation 
in  milk.  Like  Brieger's  bacillus,  the  gelatin  colonies  more  frequently  show 
a  concentric  arrangement  than  those  of  the  Bacterium  coli  commune," 


BACILLUS  g  OF  BOOKER. 

"  Found  in  one  case  of  serious  gastro-enteric  catarrh.  It  was  not  in  large 
quantity. 

'  Morphology  and  Biological  Characters. — In  morphology,  character  of 
growth  on  agar,  gelatin,  and  potato,  it  resembles  Bacterium  coli  commune. 

"  Action  on  Milk  and  Litmus  Reaction. — Milk  is  not  coagulated,  and  milk 
colored  blue  with  litmus  is  changed  to  pink  in  a  few  days,  and  holds  this 
color.  These  characteristics  distinguish  it  from  the  Bacterium  coli  com- 
mune. 

"  Gas  Production.—  Not  observed  in  milk  or  potato  cultures. 

"  AWf/f/oH  t<>  Crltttin.—  Dors  not  liquefy  gelatin.'1 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  471 

BACILLUS  k   OF  BOOKER. 

"Found  in  one  case  of  mild  dysentery,  not  in  large  quantity. 

"  Morphology. — Resembles  Bacterium  coli  commune. 

"  Growth  in  Colonies. — Gelatin:  In  plain  neutral  gelatin  the  colonies  re- 
semble those  of  Bacterium  coli  commune.  In  sugar  gelatin  the  colonies  are 
white  and  spread  extensively.  Slightly  magnified,  they  have  a  round,  dark 
centre  surrounded  by  a  yellow,  loose  zone  with  an  outer  white  rim  ;  later 
the  whole  colony  has  a  uniform  yellow  color  and  is  not  compact. 

"  Agar  :  Colonies  are  white,  round,  and  large.  Slightly  magnified,  they 
are  brownish-yellow. 

"  Stab  Cultures. — Nothing  characteristic  in  gelatin  and  agar. 

"Potato  culture  is  yellow,  dry,  and  slightly  raised,  with  well-defined  bor- 
ders. 

"  Action  on  Milk  and  Litmus  Reaction. — Milk  is  coagulated  into  a  solid 
clot  in  two  days  at  38°  C.  Milk  colored  blue  with  litmus  is  changed  to  pink 
in  twenty-four  hours. 

"  Gas  Production. — Occurs  in  milk;  not  observed  on  potato. 

"Relation  to  Gelatin. — Does  not  liquefy  gelatin.'- 

BACILLUS  k  OF  BOOKER 

"  Found  in  two  cases  of  cholera  infantum  and  one  of  catarrhal  enteritis. 

"Morphology. — Resembles  Bacterium  coli  commune. 

' '  Growth  in  Colonies. — Gelatin :  In  neutral  gelatin  the  colonies  cannot  be 
distinguished  from  those  of  Bacterium  coli  commune.  In  acid  gelatin  the 
colonies  do  not  spread  so  extensively  as  those  of  Bacterium  coli  commune, 
and  they  have  a  decided  concentric  arrangement,  a  wide  white  centre  sur- 
rounded by  a  narrow,  transparent  blue  ring,  and  outside  of  this  a  white  bor- 
der. Slightly  magnified,  the  colonies  have  an  irregular,  yellowish-brown 
centre  mottled  over  with  dark  spots  and  surrounded  by  a  light-yellow  ring 
bordered  by  a  brownish-yellow  wreath,, 

' '  Agar :  Colonies  are  large,  round,  and  bluish-white.  Slightly  magnified, 
a  light  brownish-yellow  color. 

*'  Stab  Cultures. — Gelatin :  In  sugar  gelatin  the  surface  growth  is  exten- 
sive, nearly  colorless,  and  has  a  rough,  misty  appearance.  In  the  depth  if  a 
delicate  growth.  In  plain  neutral  gelatin  the  surface  growth  is  bluish- white, 
thick,  and  not  so  extensively  spread ;  the  growth  in  the  depth  is  also  thicker. 

"  Potato  culture  is  moist,  dirty-cream  color,  has  raised  surface  and  defined 
border. 

"  Action  on  Milk. — Milk  becomes  gelatinous  in  twenty-four  hours  at  38° 
C.,  and  a  solid  clot  in  two  days.  Milk  colored  blue  with  litmus  is  changed  to 
pink  in  twenty- four  hours,  and  reduced  to  white  with  a  pink  layer  on  top  in 
two  days," 

BACILLUS  U  OF  BOOKER,, 

"Found  in  large  quantity,  but  not  the  predominating  form,  in  one  case 
of  chronic  gastro-enteric  catarrh— extremely  emaciated. 

"  Morphology.  —Resembles  Bacterium  coli  communeo 

"  Growth  in  Colonies.— Gelatin:  In  neutral  gelatin  the  colonies  are  spread 
out  and  have  a  frosty  or  ground-glass  appearance.  The  centre  is  blue  and 
border  white,  but  both  have  the  ground-glass  appearance.  Slightly  magni- 
fied, the  central  part  is  light  yellow  and  the  border  brown  with  a  rough,  fur- 
rowed surface.  In  acid  gelatin  the  white  border  is  wider  and  the  surface  is 
rougher. 

"Agar:  Colonies  are  round,  blue,  or  bluish-white,  and  spread  out.  Under 
the  microscope  they  have  a  light-yellow  color. 

"Stab  Cultures. — Gelatin:  Has  a  rough,  nearly  colorless  surface  growth, 
and  a  thick  stalk  in  the  depth  along  the  line  of  inoculation. 

' '  Agar :  Thick  white  surface  growth  with  well-developed  stalk  in  the  depth. 


472  PATHOGENIC   AEROBIC  BACILLI 

"  Action  on  Milk  and  Litmus  Reaction. — Milk  remains  liquid,  and  milk 
colored  blue  with  litmus  is  changed  to  pink. 

**  Gas  Production. — Not  observed  in  milk  or  potato  cultures. 
"  Relation  to  Gelatin. — Does  not  liquefy  gelatin. 
"Spores  have  not  been  noticed." 

Bacillus  Coli  Communis  in  Peritonitis. — The  researches  of  A. 
Frankel  show  that  Bacillus  coli  communis  may  be  obtained  in  pure 
cultures  from  the  exudate  into  the  peritoneal  cavity  in  a  considerable 
proportion  of  the  cases  of  peritonitis,  and  there  is  good  reason  for 
believing  that  in  these  cases  it  was  the  cause  of  the  inflammatory 
process.  Thirty-one  cases  were  examined  by  Frankel,  with  the  fol- 
lowing result:  Pure  cultures  of  Bacillus  coli  communis  were  obtained 
in  nine  cases ;  of  Streptococcus  (pyogenes  ?)  in  seven ;  of  Bacillus 
lactis  aerogenes  in  two ;  of  "  diplococcus  pneumonise  "  in  one  ;  of 
Staphylococcus  pyogenes  aureus  in  one.  Of  the  remaining  eleven 
cases,  seven  gave  mixed  cultures,  and  in  three  of  these  Bacillus  coli 
communis  was  the  most  abundant  species.  The  author  referred  to 
has  also  shown  that  pure  cultures  of  Bacillus  coli  communis  injected 
into  the  cavity  of  the  abdomen  of  rabbits  cause  a  typical  peritonitis. 
The  present  writer  has  frequently  obtained  the  same  result  in  experi- 
ments made  with  this  bacillus.  It  would  appear,  therefore,  that  the 
peritonitis  which  so  constantly  results  from  wounds  of  the  intestine 
is  probably  due,  to  a  considerable  extent,  to  the  introduction  of  this 
microorganism  from  the  lumen  of  the  intestine,  where  it  is  con- 
stantly found,  into  the  peritoneal  cavity,  where  the  conditions  are 
favorable  for  its  rapid  development. 

90.    BACILLUS  LACTIS  AEROGENES. 

Obtained  by  Escherich  (1886)  from  the  contents  of  the  small  intestine  of 
children  and  animals  fed  upon  milk  ;  in  smaller  numbers  from  the  faeces  of 
milk-fed  children,  and  in  one  instance  from  uncooked  cow's 
milk. 

v  Morphology. — Short  rods  with  rounded  ends,  from  1  to 

t£l  \f        2  H  in  length  and  from  0.1  to  0.5  /*  broad ;  short  oval  and 
99        spherical  forms  are  also  frequently  observed,  and,  under 
t**  f  t        certain  circumstances,  longer  rods  — 3  /' — may  be  developed : 
usually  united  in  pairs,  and  occasionally  in  chains  contain- 
ing several  elements.    In  some  of  the  larger  cells  Escherich 
has  observed  unstained  spaces,  but  was  not  able  to  obtain 
any  evidence  that  these  represent  spores. 

•heri  h  This  bacillus  stains  readily  with  the  ordinary  aniline 

colors,  but  does  not  retain  its  color  when  treated  by  Gram's 
method. 

Biological  Characters.  —  An  aerobic  (facultative  anaerobic),  non-liquefy- 
ing, non  motile  bacillus.  Does  not  form  spores.  Grows  in  various  culture 
media  at  the  room  temperature— more  rapidly  in  the  incubating  oven. 
Upon  gelatin  plates,  at  the  end  of  twenty-four  hours,  small  white  colonies 
are  developed.  Upon  the  surface  these  form  hemispherical,  soft,  shinin" 
masses  which,  examined  under  the  microscope,  are  found  to  be  homogeneous 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  473 

and  opaque,  with  a  whitish,  lustre  by  reflected  light.  The  deep  colonies  are 
spherical  and  opaque  and  attain  a  considerable  size.  In  gelatin  stick  cul- 
tures the  growth  resembles  that  of  Friedlander's  bacillus — i.e.,  an  abundant 
growth  along  the  line  of  puncture  and  a  rounded  mass  upon  the  surface, 
forming  a  "  nail-shaped  "  growth.  In  old  cultures  the  upper  portion  of  the 
gelatin  is  sometimes  clouded,  and  numerous  gas  bubbles  may  form  in  the 
gelatin.  Upon  the  surface  of  nutrient  agar  an  abundant,  soft,  white  layer 
is  developed.  Upon  old  potatoes,  in  the  incubating  oven,  at  the  end  of 
twenty-four  hours  a  yellowish -white  layer,  several  millimetres  thick,  is 
developed,  which  is  of  paste-like  consistence  and  contains  about  the  peri- 
phery a  considerable  number  of  small  gas  bubbles ;  this  layer  increases  in 
dimensions,  has  an  irregular  outline,  and  larger  and  more  numerous  gas 
bubbles  are  developed  about  the  periphery,  some  the  size  of  a  pea;  later  the 
whole  surface  of  the  potato  is  covered  with  a  creamy,  semi-fluid  mass  filled 
with  gas  bubbles.  On  young  potatoes  the  development  is  different;  a  rather 
luxuriant,  thick,  white  or  pale  yellow  layer  is  formed,  which  is  tolerably 
dry  and  has  irregular  margins ;  the  surface  is  smooth  and  shining,  and  a 
few  minute  gas  bubbles  only  are  formed  after  several  days. 

Pathogenesis. — Injections  of  a  considerable  quantity  of  a  pure  culture 
into  the  circulation  of  rabbits  and  of  guinea-pigs  give  rise  to  a  fatal  result 
within  forty-eight  hours. 

In  his  first  publication  relating  to  "  the  bacteria  found  in  the  dejecta  of 
infants  afflic ted  with  summer  diarrhoea,"  Booker  has  described  a  bacillus 
which  he  designates  by  the  letter  B,  which  closely  resembles  Bacillus  lactis 
aerogenes  and  is  probably  identical  with  it.  He  says : 

"  Summary  of  Bacillus  B. — Found  nearly  constantly  in  cholera  infan- 
tum  and  catarrhal  enteritis,  and  generally  the  predominating  form.  It 
appeared  in  larger  quantities  in  the  more  serious  cases.  It  was  not  found 
in  the  dysenteric  or  healthy  faeces.  It  resembles  the  description  of  the  Ba- 
cillus lactis  aerogenes,  but  the  resemblance  does  not  appear  sufficient  to  con- 
stitute an  identity,  and,  in  the  absence  of  a  culture  of  the  latter  for  com- 
parison, it  is  considered  a  distinct  variety  for  the  following  reasons :  Bacillus 
B  is  uniformly  larger,  its  ends  are  not  so  sharply  rounded,  and  in  all  culture 
media  long,  thick  filaments  are  seen,  and  many  of  the  bacilli  have  the  pro- 
toplasm gathered  in  the  centre,  leaving  the  poles  clear.  There  is  some 
difference  in  their  colony  growth  on  gelatin,  and  in  gelatin  stick  cultures 
bacillus  B  does  not  show  the  nail-form  growth  with  marked  end  swelling  in 
the  depth.  In  potato  cultures  the  Bacillus  lactis  aerogenes  shows  a  differ- 
ence between  old  and  new  potatoes,  while  bacillus  B  does  not  show  any 
difference. 

"Bacillus  B  possesses  decided  pathogenic  properties,  which  was  shown 
both  by  hypodermic  injections  and  feeding  with  milk  cultures." 

91.    BACILLUS    C  OF    BOOKER. 

Found  by  Booker  (1889)  in  a  case  of  cholera  infantum. 

Morphology. — Resembles  Bacillus  lactis  aerogenes  of  Escherich. 

Biological  Characters. — Resembles  Bacillus  lactis  aerogenes,  but  differs 
from  it  in  not  coagulating  milk;  the  growth  on  potato  also  is  more  luxuri- 
ant and  the  surface  is  more  thickly  covered  with  gas  bubbles. 

BACILLI   OF  JEFFRIES. 

Jeffries,  in  a  study  of  the  alvine  discharges  of  children  suffering  from 
summer  diarrhoea,  isolated  a  number  of  bacilli  resembling  Bacillus  coli 
communis  and  Bacillus  lactis  aerogenes  of  Escherich.  He  says: 

44  While  Brieger's  bacillus  and  the  lactic  acid  bacillus  of  Escherich  were 

not  found,  a  whole  group  of  species  in  growth,  form,  and  general  physiology 

closely  resembling  them  have  been  isolated.     This  group  is  represented  by 

bacilli  A,  G,  J,  K,  P,  S,  Z ;  they  seem  to  form  a  genus  ;  the  form  is  very 

33 


474 


PATHOGENIC   AEROBIC   BACILLI 


much  alike.  All  are  good  anaerobic  growers ;  all  form  gas ;  all  turn  milk 
distinctly  acid ;  and  all  closely  resemble  one  another  in  pure  cultures.  Many 
would  doubtless  class  these  altogether  as  one  species ;  but  if  species  are  to 
be  recognized  at  all,  we  must  come  to  recognizing  every  fixed  difference  as 
constituting  a  species. 

41  This  group  occurred — always  very  abundantly— in  eighteen  out  of  the 
twenty  two  cases  of  summer  diarrhoea,  and  is  also  well  represented  among 
the  kittens.  They  are,  however,  so  much  like  the  harmless  forms  found  by 
Escherich  that  they  may  for  the  present  be  laid  aside  as  of  no  specific  sig- 
nificance. They  are  also  almost  the  only  forms  tested  which  failed  to  pro- 
duce intestinal  troubles  in  kittens.  Excluding  these,  there  is  no  species,  or 
group  of  species,  left  either  generally  occurring  or  in  sufficient  numbers  to 
be  regarded  as  the  specific  pathogenic  plant  of  summer  diarrhoaa." 

92.   BACILLUS  ACIDIFORMANS. 

Obtained  by  the  writer  (1888)  from  a  fragment  of  yellow-fever  liver  pre- 
served for  forty-eight  hours  in  an  antiseptic  wrapping ;  since  obtained  from 


Fio.  150. 


Fia.  151. 
X  1,000.     From  a  photomicrograph* 


Fio.  151—  Bacillus  acidiformaos,  from  a  potato  culture. 
(Steinberg  ) 

Fio.  m.— Culture  of  Bacillus  acidiformans  in  nutrient  gelatin,  end  of  four  days  at  22°  C. 
From  a  photograph.  (Steinberg.) 

liver  preserved  in  the  same  way  from  two  comparative  autopsies— i.e.,  not 
cases  of  yellow  fever. 

Morphology.— &  short  bacillus  with  rounded  corners,  sometimes  short 
oval  in  form ;  from  H  to  3  //  in  length  and  about  1.2  #  in  breadth ;  may  grow 
out  into  filaments  of  5  to  10  >u,  or  more,  in  length;  in  some  cultures  the  short 
oval  form  predominates. 

Stains  readily  with  the  aniline  colors  usually  employed,  and  by  Gram's 
method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
/"/'":///"'.'/.  Mtn-uu*til<>,  bacillus.  Dors  not  form  spoivs.  Grows  rapidly  at 
the  room  temperature  in  the  usual  culture  media.  Grows  in  decidedly  acid 
media;  in  culture  media  containing  glycerin  or  glucose  it  produces  an  abun- 
dant evolution  of  carbon  dioxide,  and  a  volatile  acid  is  formed. 

t  does  not  liquefy  gelatin,  and  in  stick  cultures  grows  abundantly  both 
on  the  surface  and  along  the  line  of  puncture.  At  the  end  of  twenty-four 
hours,  at  22°  C.,  a  rounded  white  mass  is  formed  upon  the  surface,  resembling 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  475 

the  growth  of  Friedlander's  bacillus ;  at  the  bottom  of  the  line  of  puncture 
the  separate  colonies  are  spherical,  opaque,  and  pearl-like  by  reflected  light. 
Gas  bubbles  are  formed  in  the  gelatin.  At  the  end  of  a  week  the  surface  is 
covered  with  a  thick,  white,  semi-fluid  mass. 

In  gelatin  roll  tubes  the  superficial  colonies  are  translucent  or  opaque, 
and  circular  or  somewhat  irregular  in  outline;  by  reflected  light  they  are 
slightly  iridescent  ;  the  deep  colonies  are  spherical,  opaque,  and  homo- 
geneous. 

The  growth  upon  the  surface  of  nutrient  agar  is  abundant  and  rapid,  of 
a  shining  milk-white  color,  and  cream  like  in  consistence.  An  abundant 
development  forms  along  the  line  of  puncture  and  the  culture  medium  is 
split  up  by  gas  bubbles.  In  glycerin-agar  the  evolution  of  gas  is  very  abun- 
dant and  the  culture  medium  acquires  an  intensely  acid  reaction. 

On  potato  the  growth  is  abundant  and  rapid  at  a  temperature  of  20°  to 
30°  C.,  forming  a  thick,  semi-fluid  mass  of  a  milk-white  color. 

I  have  not  obtained  any  evidence  that  this  bacillus  forms  spores;  the 
cultures  are  sterilized  by  ten  minutes'  exposure  to  a  temperature  of  160°  F. 

When  cultivated  in  bouillon  to  which  five  per  cent  of  glycerin  has  been 
added  the  culture  medium  acquires  a  milky  opacity,  and  there  is  a  copious 
precipitate,  of  a  viscid  consistence,  consisting  of  bacilli ;  during  the  period 
of  active  development  the  surface  is  covered  with  gas  bubbles,  as  in  a  sac- 
charine liquid  undergoing  alcoholic  fermentation,  and  the  liquid  has  a  de- 
cidedly acid  reaction. 

Pathogenesis. — Pathogenic  for  rabbits  and  for  guinea  pigs  when  injected 
into  the  cavity  of  the  abdomen — one  to  two  cubic  centimetres  of  a  culture  in 
bouillon.  The  animal  usually  dies  in  less  than  twenty-four  hours.  The 
bacilli  are  found  in  the  blood  in  rather  small  numbers,  and  are  frequently 
seen  in  the  interior  of  the  leucocytes.  The  spleen  is  enlarged,  the  liver 
normal,  the  intestine  usually  hyperaemic. 

93.    BACILLUS  CUNICULICIDA  HAVANIENSIS. 

Obtained  by  the  writer  (1889)  from  the  contents  of  the  intestine  of  yellow- 
fever  cadavers,  and  also  from  fragments  of  yellow-fever  liver  preserved  for 
forty-eight  hours  in  an  antiseptic  wrap- 
ping— my  bacillus  a?,  Havana,  1889. 

Morphology. — This  bacillus  resembles 
the  colon  bacillus  in  form,  but  is  some- 
what larger,  from  2  to  4  //  in  length  and 
from  0.8  to  1  /*  in  diameter  ;  sometimes 
associated  in  pairs ;  may  grow  out  into 
short  filaments — not  common.  The  ends 
of  the  rods  are  rounded,  and  under  cer- 
tain circumstances  vacuoles  are  seen  at 
the  extremities,  especially  in  potato  cul- 
tures. 

Stains  quickly  with  the  aniline  colors 
usually  employed,  and  also  by  Gram's 
method. 

Biological  Characters. — An  aerobic 
and  facultative  anaerobic,  non-lique- 
fying bacillus.  Under  certain  circum-  FIG.  152.— Bacillus  cuniculicida  Havani- 
stances  may  exhibit  active  movements,  ensis,  from  a  single  colony  in  nutrient  gela- 
but  is  usually  motionless.  tin-  *  ^m-  Frona  a  photomicrograph. 

A  very  curious  thing  with  reference    (Sternberg.) 
to  this  bacillus  is  that  it  presented  ac- 
tive movements  in  my  cultures  made  directly  from  yellow-fever  cadavers, 
but  that  these  movements  were  not  constant,  and  that  since  my  return  to 
Baltimore  I  have  not,  as  a  rule,  observed  active  movements  in  cultures  from 
the  same  stock,  which,  however,  preserved  their  pathogenic  power  and  other 


476 


PATHOGENIC   AEROBIC  BACILLI 


characters.  In  Havana  these  movements  were  usually  not  observed  in  all 
the  bacilli  in  a  field  under  observation,  but  one  and  another  would  start  from 
a  quiescent  condition  on  an  active  and  erratic  course ;  sometimes  spinning 
actively  upon  its  axis,  and  again  shooting  across  the  field  as  if  propelled  by 
a  nagellum. 

My  notes  indicate  that  cultures  passed  through  the  guinea-pig  are  more 
apt  to  be  motile. 

In  gelatin  stick  cultures  the  growth  of  bacillus  x  resembles  that  of  the 
colon  bacillus,  but  the  colonies  at  the  bottom  of  the  line  of  puncture  are 
more  opaque  and  not  of  a  clear  amber  color  like  that  of  colonies  of  the  colon 
bacillus.  Upon  the  surface  the  growth  is  thicker  than  that  of  the  colon 
bacillus,  and  forms  a  milk-white,  soft  mass. 

The  colonies  in  gelatin  Esmarch  roll  tubes  vary  considerably  at  different 
times.  Deep  colonies  are  usually  spherical,  homogeneous,  light  brown  in 
color,  and  more  opaque  than  the  similar  colonies  of  the  colon  bacillus.  At 
the  end  of  a  few  days  the  deep  colonies  become  quite  opaque,  and  may  be 
lobate,  like  a  mulberry,  or  coarsely  granular ;  sometimes  the  deep  colonies 
have  an  opaque  central  portion  surrounded  by  a  transparent  marginal  zone. 

In  old  gelatin  roll  tubes  these  deep  colonies  form  opaque  white  hemi- 


Fio.  163.  FIG.  154. 

Fig.  158.— Bacillus  cuniculicida  Havaniensis;  colonies  in  gelatin  roll  tube,  third  day  at  20°  C. 
X  6.  From  a  photograph.  (Sternberg.) 

Fio.  154.— Bacillus  cuniculicida  Havaniensis ;  colonies  in  gelatin  roll  tube,  end  of  forty-eight 
hours,  x  10.  From  a  photograph.  (Sternberg.) 

spheres  projecting  from  the  surface  of  the  dried  culture  medium,  and  little 
tufts  of  acicular  crystals  are  sometimes  observed  to  project  from  the  side  of 
such  old  colonies. 

The  superficial  colonies  are  circular  or  irregular  in  outline,  with  trans- 
puiviit  margins  ;uul  an  opaque  central  portion,  sometimes  corrugated.  They 
are  finely  granular  and  iridescent  by  reflected  light,  and  of  a  milk-white 
color;  by  transmitted  light  they  have  a  brownish  color.  Young  colonies 
closely  resemble  those  of  the  colon  bacillus.  This  bacillus  grows  well  at  a 
temperature  of  20°  C.  (68°  F.),  but  more  rapidly  and  luxuriantly  at  a  higher 
temperature— 30°  to  35°  C. 

It  grows  well  in  agar  cultures,  and  especially  inglycerin-agar,'m  which 
it  produces  some  gas  and  an  acid  reaction.  The  growth  on  the  surface 
of  glycerm-agar  cultures  is  white,  cream-like  in  consistence,  and  quite  abun- 
dant. 

It  grows  well  in  an  agar  or  gelatin  medium  made  acid  by  the  addition  of 
0.2  per  cent  (1:  500)  of  hydrochloric  acid. 


NOT   DESCRIBED   IN  PREVIOUS   SECTIONS.  477 

In  cocoanut  water  it  multiplies  rapidly,  producing  a  milky  opacity  of  the 
previously  transparent  fluid,  an  acid  reaction,  and  an  evolution  of  carbon 
dioxide. 

On  potato  it  produces  a  thick  layer,  which  may  cover  the  entire  surface 
in  three  or  four  days,  and  which  has  a  dirty-white,  cream- white,  or  pinkish- 
white  color  and  cream-like  consistence.  The  growth  upon  potato  varies  at 
different  times,  evidently  owing  to  differences  in  the  potato. 

When  stained  preparations  are  examined  with  the  full  light  of  the  Abbe 
condenser  the  ends  of  some  of  the  rods  appear  to  be  cut  away,  leaving  a  con- 
cave extremity ;  but  by  using  a  small  diaphragm  to  obtain  definition  it  will 
be  seen  that  the  cell  wall  extends  beyond  the  stained  portion  of  the  rod  and 
includes  what  appears  to  be  a  vacuole.  There  is  no  reason  to  believe  that 
this  appearance  is  due  to  the  presence  of  an  end  spore,  for  the  supposed 
vacuole  is  not  refractive,  as  a  spore  would  be,  and  my  experiments  on  the 
thermal  death-point  of  this  bacillus  indicate  that  it  does  not  form  spores. 
Cultures  are  sterilized  by  exposure  for  ten  minutes  to  a  temperature  of  160° 
F.  (71.2°  C.). 

Pathogenesis. — Very  pathogenic  for  rabbits  when  injected  into  the  cavity 
of  the  abdomen.  Injections  of  a  small  quantity  of  a  pure  culture  into  the 
ear  vein  or  subcutaneously  generally  give  a  negative  result.  Injections  of 
from  one  to  five  cubic  centimetres  of  a  culture  in  bouillon,  blood  serum,  or 
agua  coco,  into  the  cavity  of  the  abdomen,  frequently  prove  fatal  to  rabbits 
in  a  few  hours — two  to  six. 

The  negative  results  obtained  in  injecting  cultures  beneath  the  skin  or 
into  the  ear  vein  of  rabbits  show  that  this  bacillus  does  not  induce  a  fatal 
septicaemia  in  these  animals,  and  the  fatal  result  when  injections  are  made 
into  the  peritoneal  cavity  does  not  appear  to  be  due  to  an  invasion  of  the 
blood,  but  rather  to  the  local  effect  upon  the  peritoneum,  together  with  the 
toxic  action  of  the  chemical  products  resulting  from  its  growth. 

It  is  true  that  I  have  always  been  able  to  recover  the  bacillus  from  the 
liver,  or  from  blood  obtained  from  one  of  the  cavities  of  the  heart,  even  in 
animals  which  succumb  within  a  few  hours  to  an  injection  made  into  the 
cavity  of  the  abdomen.  But  the  direct  examination  of  the  blood  shows  that 
the  bacilli  are  present  in  very  small  numbers,  and  leads  me  to  believe  that 
the  bacillus  does  not  multiply,  to  any  considerable  extent  at  least,  in  the 
circulating  fluid. 

The  spleen  is  not  enlarged,  as  is  the  case  in  anthrax,  rabbit  septicaemia, 
and  other  diseases  in  which  the  pathogenic  microorganism  multiplies  abun- 
dantly in  the  blood. 

On  the  other  hand,  there  is  evidence  of  local  inflammation  in  the  peri- 
toneal cavity.  When  death  occurs  within  a  few  hours  the  peritoneum  is 
more  or  less  hyperaemic  and  there  is  a  considerable  quantity  of  straw-colored 
fluid  in  the  cavity  of  tbe  abdomen.  When  the  animal  lives  for  twenty 
hours  or  more  there  is  a  decided  peritonitis  with  a  fibrinous  exudation  upon 
the  surface  of  the  liver  and  intestine.  Usually  the  liver,  in  animals  which 
die  within  twenty-four  hours,  is  full  of  blood,  rather  soft,  and  dark  in  color. 
In  a  single  instance  I  found  the  liver  to  be  of  a  light  color  and  loaded  with 
fat. 

The  rapidly  fatal  effect  in  those  cases  in  which  I  have  injected  two  or 
more  cubic  centimetres  of  a  culture  into  the  cavity  of  the  abdomen  has  led 
me  to  suppose  that  death  results  from  the  toxic  effects  of  a  ptomaine  con- 
tained in  the  culture  at  the  time  of  injection.  The  symptoms  also  give  sup- 
port to  this  supposition.  The  animal  quickly  becomes  feeble  and  indisposed 
to  move,  and  some  time  before  death  lies  helpless  upon  its  side,  breathing 
regularly,  but  is  too  feeble  to  get  up  on  its  feet  when  disturbed.  Death  some- 
times occurs  in  convulsions,  but  more  frequently  without— apparently  from 
heart  failure. 

Pathogenic  also  for  guinea-pigs  when  injected  into  the  cavity  of  the 
abdomen,  but  death  does  not  occur  in  so  short  a  time — eighteen  to  twenty 
hours.  Subcutaneous  injections  of  one-half  to  one  cubic  centimetre  gave  a 


478  PATHOGENIC   AEROBIC   BACILLI 

negative  result  in  eleven  out  of  thirteen  guinea-pigs  inoculated — two  died 
within  twenty-four  hours. 

94.    BACILLUS   LEPORIS   LETHALIS. 

Obtained  by  Dr.  Paul  Gibier  (1888)  from  the  contents  of  the  intestine  of 
yellow-fever  patients ;  also  by  the  writer  from  the  same  source  (1888,  1889) 
in  exceptional  cases  and  in  comparatively  small  numbers.  Named  and  de- 
scribed by  present  writer. 

Morphology. — Bacilli  with  rounded  ends,  from  1  to  3  UL  in  length  and 
about  0.5  u  in  breadth.  The  length  may  vary  in  the  same  culture  from  a 
short  oval  to  rods  which  are  two  or  three  times  as  long  as  broad,  or  it  may 
grow  out  into  flexible  filaments  of  considerable  length.  In  recent  cultures 
the  bacilli  are  frequently  united  in  pairs. 

Stains  readily  with  the  aniline  colors  usually  employed.  In  cultures 
which  are  several  days  old,  or  in  recent  cultures  when  the  stained  prepara- 
tion is  washed  in  alcohol,  the  ends  of  the  rods  are  commonly  more  deeply 
stained  than  the  central  portion — "  end  staining";  and  in  old  cultures  some 
of  the  bacilli  are  very  faintly  stained. 

Biological  Characters. — Anaerobic,  liquefying,  actively  motile  bacillus. 
Does  not  form  spores. 

In  gelatin  stick  cultures,  at  the  end  of  twenty-four  hours  at  a  tempe- 
rature of  20°  to  22°  C.,  there  is  an  abundant  development  along  the  line  of 
puncture  and  commencing  liquefaction  at  the  surface.  Later  the  liquefaction 
is  funnel-shaped,  and  there  is  an  opaque  white  central  core  along  the  line 
of  puncture,  with  liquefied  gelatin  around  it.  Liquefaction  progresses  most 
rapidly  at  the  surface,  and  in  the  course  of  three  or  four  days  the  upper  por- 
tion of  the  gelatin  for  a  distance  of  half  an  inch  or  more  is  completely  lique- 
fied, and  an  opaque  white  mass,  composed  of  bacilli,  rests  upon  the  surface 
of  the  unliquefied  portion. 

In  gelatin  roll  tubes  the  young  colonies  upon  the  surface  are  transparent 
and  resemble  somewhat  small  fragments  of  broken  glass;  later  liquefaction 
occurs  rapidly.  Deep  colonies  in  gelatin  roll  tubes,  or  at  the  bottom  of  stick 
cultures,  are  spherical,  translucent,  and  of  a  pale  straw  color. 

Upon  the  surface  of  nutrient  agar  it  grows  rapidly,  forming  a  rather  thin, 
translucent,  shining,  white  layer,  which  covers  the  entire  surface  at  the  end 
of  two  or  three  days  at  a  temperature  of  20°  C. 

Upon  potato  the  growth  is  rapid  and  thin,  covering  the  entire  surface, 
and  is  of  a  pale-yellow  color. 

This  bacillus  grows  at  a  comparatively  low  temperature,  and  its  vitality 
is  not  destroyed  by  exposure  for  an  hour  and  a  half  in  a  freezing  mixture  at 
15°  C.  below  zero  (5°  F.). 

Decided  growth  occurred  in  a  stick  culture  in  gelatin  exposed  in  Balti- 
more during  the  month  of  January  in  an  attic  room.  During  the  twenty- 
two  days  of  exposure  the  highest  temperature,  taken  at  9  A.M.  each  day, 
was  11°  C.,  and  the  lowest  2°  C.  At  a  temperature  of  16°  to  20°  C.  develop- 
ment in  a  favorable  culture  medium  is  rapid. 

There  is  no  evidence  that  this  bacillus  forms  spores ;  cultures  are  sterilized 
by  exposure  to  a  temperature  of  60°  C.  for  ten  minutes. 

Coagulated  blood  serum  is  liquefied  by  this  bacillus.  It  retains  its  vitality 
for  a  long  time  in  old  cultures,  having  grown  freely  when  replanted  at  the 
end  of  a  year  from  a  hermetically  sealed  tube  containing  a  pure  culture  ia 
blood  serum. 

Pathogenefds.—  This  bacillus  is  very  pathogenic  for  rabbits  when  injected 
into  the  cavity  of  the  abdomen  in  quantities  of  one  cubic  centimetre  or  more ; 
it  is  less  pathogenic  for  guinea-pigs,  and  is  not  pathogenic  for  white  rats 
when  iniected  subcutaneously.  Gelatin  cultures  seem  to  possess  more  in- 
tense pathogenic  power  than  bouillon  cultures,  and  cultures  from  the  blood 
of  an  animal  recently  dead  as  the  result  of  an  inoculation  are  more  potent 


NOT   DESCRIBED   IN  PREVIOUS   SECTIONS.  479 

than  those  from  my  original  stock  which  had  not  been  passed  through  a 
susceptible  animal. 

The  mode  of  death  in  rabbits  is  quite  characteristic.  A  couple  of  hours 
after  receiving  in  the  cavity  of  the  abdomen  two  or  three  cubic  centimetres 
of  a  liquefied  gelatin  culture  the  animal  becomes  quiet  and  indisposed  to  eat 
or  move  about.  Soon  after  it  becomes  somnolent,  the  head  drooping  for- 
ward and  after  a  time  restiug  between  the  front  legs,  with  the  nose  on  the 
floor  of  its  cage.  It  can  be  roused  from  this  condition,  and  raises  its  head  in 
an  indifferent  and  stupid  way  when  pushed  or  shaken,  but  soon  drops  off 
again  into  a  profound  sleep.  Frequently  the  animals  die  in  a  sitting  posi- 
tion, with  their  nose  resting  upon  the  floor  of  the  cage  between  the  front 
legs.  I  have  not  seen  this  lethargic  condition  produced  by  inoculations  with 
any  other  microorganism.  Convulsions  sometimes  occur  at  the  moment  of 
death. 

The  time  of  death  depends  upon  the  potency  of  the  culture  and  its  quan- 
tity as  compared  with  the  size  of  the  animal.  From  three  to  four  cubi« 
centimetres  of  a  liquefied  gelatin  culture  usually  kill  a  rabbit  in  from  three 
to  seven  hours. 

The  rapidity  with  which  death  occurs  when  a  considerable  quantity  of  a 
liquefied  gelatin  culture  is  injected  into  the  cavity  of  the  abdomen,  and  the 
somnolence  which  precedes  death,  give  rise  to  the  supposition  that  t^e  lethal 
effect  is  due  to  the  presence  of  a  toxic  chemical  substance  rather  than  to  a 
multiplication  of  the  bacillus  in  the  body  of  the  animal.  And  this  view  is 
supported  by  the  fact  that  animals  frequently  recover  when  the  dose  admin- 
istered is  comparatively  small  and  especially  when  it  is  injected  subcuta- 
neously. 

In  all  cases  in  which  death  occurs,  even  when  but  a  few  hours  have 
elapsed  since  the  inoculation  was  made,  I  have  recovered  the  bacillus  in 
cultures  made  from  blood  obtained  from  the  heart  or  the  interior  of  the 
liver,  and,  as  stated,  these  cultures  appear  to  have  a  greater  virulence  than 
those  not  passed  through  the  rabbit. 

In  sections  of  the  liver  and  kidney  stained  with  Loffler's  solution  of 
methylene  blue  the  bacilli  are  seen,  and  are  often  in  rather  long-jointed  fil- 
aments. 


95.   BACILLUS  PYOCYANEUS.    vxj.  ^fc   Jy^  ^  2.  "]  ] 

Synonyms.  —  Bacillus  of  green  pus  ;  Microbe  du  pus  bleu;  Bacil- 
len  des  grunblauen  Eiters  ;  Bacterium  aeruginosum. 

Obtained  by  Gessard  (1882)  from,  pus  having  a  green  or  blue 
color,  and  since  carefully  studied  by  Gessard,  Charrin,  and  others. 
This  bacillus  appears  to  be  a  widely  distributed 
saprophyte,  which  is  found  occasionally  in  the 
purulent  discharges  from  open  wounds,  and  some- 
times in  perspiration  and  serous  wound  secretions 
(Gessard)  .    The  writer  obtained  it,  in  one  instance, 
FIG.  155.  —  Bacillus      in  cultures  from  the  liver  of  a  yellow-fever  cada- 
X7°°'      ver  (Havana,  1888). 

Morphology.  —  A  slender  bacillus  with  rounded 
ends,  somewhat  thicker  than  the  Bacillus  murisepticus  and  of  about 
the  same  length  (Fliigge)  ;  frequently  united  in  pairs,  or  chains  of  four 
to  six  elements;  occasionally  grows  out  into  filaments. 

Biological  Characters.  —  An  aerobic,  liquefying,  motile  bacil- 
lus.    Grows  readily  in  various  culture  media  at  the  room  tempera- 


480  PATHOGENIC   AEROBIC   BACILLI 

ture — more  rapidly  in  the  incubating  oven.  Is  a  facultative  anae- 
robic (Frankel).  Does  not  form  spores.  The  thermal  death-point, 
as  determined  by  the  writer,  is  56°  C.,  the  time  of  exposure  being  ten 
minutes.  In  gelatin  plate  cultures  colonies  are  quickly  developed, 
which  give  to  the  medium  a  fluorescent  green  color  ;  at  the  end  of 
two  or  three  days  liquefaction  commences  around  each  colony,  and 
usually  the  gelatin  is  completely  liquefied  by  the  fifth  day.  Before 
liquefaction  commences  the  deep  colonies,  under  a  low  power,  appear 
as  spherical,  granular  masses,  with  a  serrated  margin,  and  have  a 
yellowish-green  color ;  the  superficial  colonies  are  quite  thin  and 
finely  granular  ;  at  the  centre,  where  they  are  thickest,  they  have  a 
greenish  color,  which  fades  out  towards  the  periphery. 

In  stick  cultures  in  nutrient  gelatin  development  is  most  abun- 
dant near  the  surface,  and  causes  at  first  liquefaction  in  the  form 
of  a  shallow  funnel ;  later  the  liquefied  gelatin  is  separated  from 
that  which  is  not  liquefied  by  a  horizontal  plane,  and  a  viscid,  yel- 
lowish-white mass,  composed  of  bacilli,  accumulates  upon  this  sur- 
face, which  gradually  has  a  lower  level  as  liquefaction  progresses  ; 
the  transparent,  liquefied  gelatin  above  is  covered  with  a  delicate, 
yellowish-green  film,  and  the  entire  medium  has  a  fluorescent  green 
color.  Upon  nutrient  agar  a  rather  abundant,  moist,  greenish-white 
layer  is  developed,  and  the  medium  acquires  a  bright  green-color, 
which  subsequently  changes  to  olive  green.  Upon  potato  a  viscid 
or  rather  dry,  yellowish-green  or  brown  layer  is  formed,  and  the 
potato  beneath  and  immediately  around  the  growth  has  a  green  color 
when  freely  exposed  to  the  air  or  to  the  vapors  of  ammonia.  In  milk 
the  casein  is  first  precipitated  and  then  gradually  dissolved,  while  at 
the  same  time  ammonia  is  developed.  The  green  pigment  is  formed 
only  in  the  presence  of  oxygen;  it  is  soluble  in  chloroform  and  may 
be  obtained  from  a  pure  solution  in  long,  blue  needles ;  acids  change 
the  blue  color  to  red,  and  reducing  substances  to  yellow.  According 
to  Ledderhose,  it  is  an  aromatic  compound  resembling  anthracene, 
and  is  not  toxic.  According  to  Gessard's  latest  researches  (1890),  two 
different  pigments  are  produced  by  this  bacillus,  one  of  a  fluorescent 
green  and  the  other — pyocyanin — of  a  blue  color.  Cultivated  in  egg 
albumin  the  fluorescent  green  pigment,  which  changes  to  brown 
with  time,  is  alone  produced.  In  bouillon  and  in  media  containing 
peptone  or  gelatin  both  pigments  are  formed,  and  the  pyocyanin 
may  be  obtained  separately  by  dissolving  it  in  chloroform.  In  an 
alkaline  solution  of  peptone  (two  per  cent)  to  which  five  per  cent  of 
glycerin  has  been  added  the  blue  pigment  alone  is  formed. 

Pathogenesis. — The  experiments  of  Ledderhose,  Bouchard,  and 
others  show  that  this  bacillus  is  pathogenic  for  guinea-pigs  and  rab- 
bits. Subcutaneous  or  intraperitoneal  injections  of  recent  cultures — 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  481 

one  cubic  centimetre  or  more  of  a  culture  in  bouillon — usually  cause 
the  death  of  the  animal  in  from  twelve  to  thirty-six  hours.  An  ex- 
tensive inflammatory  oedema  and  purulent  infiltration  of  the  tissues 
result  from  subcutaneous  inoculations,  and  a  sero-fibrinous  or  puru- 
lent peritonitis  is  induced  by  the  introduction  of  the  bacillus  into  the 
peritoneal  cavity.  The  bacillus  is  found  in  the  serous  or  purulent 
fluid  in  the  subcutaneous  tissues  or  abdominal  cavity,  and  also  in  the 
blood  and  various  organs,  from  which  it  can  be  recovered  in  pure 
cultures,  although  not  present  in  great  numbers,  as  is  the  case  in 
the  various  forms  of  septicaemia  heretofore  described.  When  smaller 
amounts  are  injected  subcutaneously  the  animal  usually  recovers 
after  the  formation  of  a  local  abscess,  and  it  is  subsequently  immune 
when  inoculated  with  doses  which  would  be  fatal  to  an  unprotected 
animal.  Immunity  may  also  be  secured  by  the  injection  of  a  con- 
siderable quantity  of  a  sterilized  culture.  Bouchard  has  also  pro- 
duced immunity  in  rabbits  by  injecting  into  them  the  filtered  urine 
of  other  rabbits  which  had  been  inoculated  with  a  virulent  culture  of 
the  bacillus.  It  has  been  shown  by  Bouchard,  and  by  Charrin  and 
Guignard,  that  in  rabbits  which  have  been  inoculated  with  a  culture 
of  the  anthrax  bacillus  a  fatal  result  may  be  prevented  by  soon  after 
inoculating  the  same  animals  with  a  pure  culture  of  the  Bacillus 
pyocyaneus.  The  experiments  of  Woodhead  and  Wood  indicate  that 
the  antidotal  effect  is  due  to  chemical  products  of  the  growth  of  the 
bacillus,  and  not  to  an  antagonism  of  the  living  bacterial  cells.  They 
were  able  to  obtain  similar  results  by  the  injection  of  sterilized  cul- 
tures of  Bacillus  pyocyaneus,  made  soon  after  infection  with  the 
anthrax  bacillus. 

Schimmelbusch  (1894)  reports  that  in  researches  made  by  Muh- 
sam  this  bacillus  was  found  in  the  axilla,  the  anal  region,  or  the  in- 
guinal fold  in  fifty  per  cent  of  the  healthy  individuals  examined. 
Its  presence  in  wounds  greatly  delays  the  process  of  repair  and  may 
give  rise  to  a  general  depression  of  the  vital  powers  from  the  ab- 
sorption of  its  toxic  products.  Schimmelbush  states  that  a  physician 
injected  0.5  cubic  centimetre  of  sterilized  (by  heat)  culture  into  his 
forearm.  That  as  a  result  of  this  injection,  after  a  few  hours  he  had 
a  slight  chill,  followed  by  fever,  which  at  the  end  of  twelve  hours 
reached  38.8°  ;  an  erysipelatous  -  like  swelling  of  the  forearm  oc- 
curred, and  the  glands  in  the  axilla  were  swollen  and  painful.  Re- 
covery occurred  without  the  formation  of  an  abscess.  Buchner  has 
related  a  similar  case. 

Krannhals  (1894)  refers  to  seven  cases  in  which  a  general  pyocy- 
aneus infection  in  man  was  found,  and  adds  an  eighth  from  his  own 
experience.  In  this  the  Bacillus  pyocyaneus  was  obtained,  post  mor- 

34 


482  PATHOGENIC   AEROBIC   BACILLI 

tern,  from  green  pus  in  the  pleural  cavity,  from  serum  in  the  peri- 
cardial  sac,  and  from  the  spleen,  in  pure  culture. 

Martha,  Gruber,  Maggiora,  Gradenigo,  Kossel,  and  Rohrer  have 
reported  cases  in  which  the  Bacillus  pyocyaneus  has  been  obtained  in 
pure  cultures  from  pus  obtained  from  the  tympanic  cavity  in  middle- 
ear  disease.  Kossel  (1894)  relates  several  cases  in  his  own  experience 
which  led  him  to  the  conclusion  that,  in  children,  the  Bacillus  pyocy- 
aneus, through  general  blood  infection  or  indirectly  through  the 
absorption  of  its  toxic  products,  may  be  the  cause  of  death. 

The  following  varieties  of  this  bacillus  have  been  described  by 
bacteriologists : 

BACILLUS  PYOCYANEUS  /?  (P.   Ernst). 

Found  in  pus  from  bandages  colored  green. 

Morphology.—  Slender  bacilli  from  2  to  4 //long — occasionally  5  to  6  n — 
and  from  0.5  to  0.75 //broad;  sometimes  united  in  pairs,  or  chains  of  three 
elements. 

Biological  Characters. — An  aerobic,  liquefying,  actively  motile,  chro- 
mogenic  bacillus.  Produces  a  yellowish-green  pigment;  when  old  cul- 
tures are  shaken  up  with  chloroform  and  this  is  allowed  to  stand,  three 
layers  are  formed — an  upper,  clouded,  dirty-yellow  layer  ;  below  this  is  a 
milky,  pale-green  layer  ;  and  at  the  bottom  a  transparent,  azure-blue  layer. 
Spore  formation  has  not  been  demonstrated.  Grows  in  the  usual  culture 
media  at  the  room  temperature— more  rapidly  at  35°  C.  Upon  gelatin  plates 
colonies  are  formed  resembling  those  of  the  well-known  Bacillus  pyocyaneus, 
but  liquefaction  is  more  rapid.  In  gelatin  stick  cultures  funnel-shaped 
liquefaction  occurs  at  the  upper -part  of  the  line  of  puncture  by  the  third 
day,  and  progresses  more  rapidly  than  is  the  case  with.  Bacillus  pyocyaneus 
under  the  same  circumstances  ;  on  the  fifth  day  a  bluish-green  color  is  de- 
veloped; by  the  twelfth  day  liquefaction  has  obliterated  the  entire  line  of 
growth  and  extends  to  the  margins  of  the  tube;  the  liquefied  gelatin  for  a 
depth  of  about  one  centimetre  has  a  dark  emerald-green  color,  and  a  film 
consisting  of  bacilli  is  seen  upon  the  surface.  Upon  the  surface  of  agar  a 
flat,  greenish- white,  dry  layer  is  formed  along  the  line  of  inoculation,  and 
the  agar  around,  at  the  end  of  a  week,  acquires  a  bluish-green  color.  Upon 
potato,  at  the  end  of  three  days,  an  abundant  dry  layer  of  a  fawn  brown 
color  has  developed  ;  this  is  surrounded  by  a  pale-green  coloration  of  the 
potato,  and  at  points  where  the  surface  is  fissured,  an  intense  dark-green 
color  is  developed;  the  growth  on  potato  has  a  more  or  less  wrinkled  appear- 
ance ;  when  one  of  the  fawn-colored  colonies  is  touched  with  the  platinum 
needle,  the  point  touched,  at  the  end  of  two  to  five  minutes,  acquires  an  in- 
tense dark  leaf-green  color,  which  reaches  its  maximum  intensity  in  about 
ten  minutes,  and  has  faded  out  again  at  the  end  of  half  an  hour.  Ernst  con- 
siders this  "chameleon  phenomenon"  the  most  characteristic  distinction 
between  the  bacillus  under  consideration  and  Bacillus  pyocyaneus.  In  milk 
a  green  color  is  developed  at  the  surface,  the  casein  is  precipitated  and  sub- 
sequently peptonized. 

Bacillus  pyocyaneus  peri  car  ditidis.  Found  by  H.  C.  Ernst  in 
fluid  obtained  by  tapping  the  pericardial  sac  of  a  man  aged  forty- 
seven  years.  Fluid  was  drawn  from  the  pericardial  sac  on  four  dif- 
ferent occasions.  The  man  subsequently  "eloped."  Ernst  gives  the 
following  description  of  this  bacillus : 


NOT   DESCRIBED   IN   PREVIOUS  SECTIONS.  483 

ORIGIN. — Pericardial  fluid,  containing  also  bacilli  of  tuberculosis. 

FORM  AND  ARRANGEMENT. — Small  straight  bacilli,  with  rounded  ends, 
three  or  four  times  as  long  as  broad,  and  on  most  media  slightly  larger  than 
the  Bacillus  pyocyaneus  of  Gessard,  occurring  -within  the  cells  in  the  origi- 
nal fluid,  and  sometimes  showing  two  or  three  end  to  end,  but  never  observed 
in  long  chains. 

MOTILITY. — Actively  motile  in  hanging-drop  culture.  No  cilia  or  flagel- 
la  have  been  demonstrated. 

GROWTH — Gelatin  :  Plates. — Colonies  appear  at  the  end  of  thirty-six  to 
forty-eight  hours  as  fine  white  points  in  the  interior,  and  upon  the  surface  of 
the  medium ;  edges  are  sharply  defined  ;  soon  there  appears  a  circular  zone 
of  liquefaction,  finally  passing  through  the  stratum  of  the  medium  with 
the  colony  at  the  bottom.  Under  a  low  power  the  centre  of  the  colony  may 
be  of  a  brownish  color.  On  the  second  day  a  greenish  tinge  may  be  seen 
about  the  individual  colonies  on  the  surface  which  spreads  through  the 
entire  medium.  The  plates  may  always  be  distinguished  from  those  of  the 
Bacillus  pyocyaneus  of  Gessard  by  the  bluish-green  when  contrasted  with 
the  yellowish-green  color  of  this  latter. 

Gelatin:  Needle  Cultures. — At  the  end  of  twenty-four  hours  a  small, 
saucer-shaped  depression  of  liquefaction  at  upper  end  of  needle  track,  which 
gradually  spreads  and  deepens  until  the  liquefaction  extends  straight  across 
the  tube,  and  about  half-way  down  the  needle  track.  A  bluish-green  fluores- 
cence appears  about  the  liquefied  portion  at  the  very  upper  part  of  the  gela- 
tin, later  changing  into  a  yellowish  green.  The  colony  is  deposited  as  a 
yellowish,  heavy  sediment  at  the  bottom  of  the  liquefied  portion,  the  upper 
part  of  which  is  clear.  A  small,  whitish  growth  occurs  along  the  remainder 
of  the  needle  track.  Old  cultures,  in  which  a  certain  amount  of  evapora- 
tion has  occurred,  assume  a  very  dark  greenish-black  color. 

Agar-agar. — Along  the  needle  track  appears  aflat,  dry  colony  of  a  dirty 
grayish- white  color  spreading  out  upon  each  side  of  the  needle  track  and 
growing  at  first  upon  the  surface  of  the  water  of  condensation,  later  depos- 
iting a  white  sediment  at  the  bottom.  From  the  first  there  may  be  detected, 
by  reflected  light,  a  metallic  lustre  on  the  surface  of  the  colony  in  places, 
which  metallic  sheen  later  spreads  over  the  whole  colony  and  furnishes  a 
marked  differentiating  point.  In  addition  to  this,  within  twenty-four  to 
forty-eight  hours  at  37°  C.,  there  appears  a  green  fluorescence  throughout 
the  whole  of  the  medium,  which  increases  slowly  to  a  marked  bluish-green 
color,  and  never  assumes  the  nut-brown  of  the  Bacillus  pyocyaneus  of 
Gessard  upon  the  same  medium.  The  colony  is  not  especially  viscid. 

Potato. — There  appears  a  reddish-brown  colony  along  the  needle  track, 
elevated  and  moist,  confined  to  the  line  of  the  needle.  It  presents  no  change 
of  color  upon  touching  with  the  needle,  but  certain  specimens  (as  do  some  of 
the  Bacillus  pyocyaneus)  develop  later  a  heavy  green  color  extending  over 
the  whole  surface  of  the  potato,  which  later  changes  almost  to  black. 

Bouillon.— Twenty-four  hours  at  37°  C.  gives  a  growth,  especially  on  the 
surface,  which  is  a  wrinkled  scum  ;  no  cloudiness  of  the  bouillon,  and  a  very 
faint  greenish  fluorescence  one  centimetre  below  the  surface.  At  this  time 
it  differs  from  the  Bacillus  pyocyaneus  of  Gessard,  in  that  the  latter  shows 
cloudiness  of  the  medium  all  through.  Later  the  same  cloudiness  appears  in 
bouillon  cultures  of  this  new  bacillus,  together  with  a  whitish  sediment  de- 
posited at  the  bottom  of  the  tube,  and  then  the  cultures  are  indistinguishable 
from  each  other.  The  same  changes,  but  slower,  occur  at  room  tempera- 
ture. 

Peptone.~One,  3.5,  and  six-per-cent  solution.  Twenty-four  hours  at  37° 
C.  gives  a  faint  bluish  tinge  at  upper  edge  of  medium  with  very  faint  cloudi- 
ness ;  later  (in  one  or  two  weeks)  there  forms  a  marked  scum  upon  the  sur- 
face that  is  difficult  to  break  up  by  shaking,  and  the  whole  medium  assumes 
a  grass-green  color  of  more  or  less  intensity,  and  not  seen  on  other  similar 
bacilli.  The  shape  and  size  of  the  organism,  under  the  microscope,  differ 


484  PATHOGENIC  AEROBIC  BACILLI 

very  markedly  in  this  medium  from  any  other  bacilli  examined.  The  same 
changes  are  to  be  seen  at  room  temperature,  but  more  slowly. 

Egg- Albumin:  Plain. — Twenty  four  hours  at  37°  C.,  yellowish-white, 
very  prof  use  growth  all  along  the  needle  track  ;  yellowish-green  spreading 
out  liom  it  almost  to  sides  of  tube,  and  in  the  condensation  water  as  well. 
The  growth  has  no  especial  distinguishing  characteristics.  Irregular  lique- 
faction occurs,  but  the  growth  at  no  time  differs  in  any  marked  way  from 
other  varieties  of  the  Bacillus  pyocyaneus. 

Blood  Serum. — Twenty-four  hours  at  37°  C.  shows  flat,  moist  colony 
with  bluish-green  fluorescence  in  its  neighborhood.  Liquefaction  begins 
early  and  goes  on  slowly  until  complete  in  from  one  to  two  weeks,  with  an 
increasing  intensity  of  color  which  becomes  markedly  blue,  and  eventually 
almost  black. 

Milk.— Behaves  as  do  the  other  bacteria. 

BEHAVIOR  TO  TEMPERATURE. — Grows  at  15°-25°  C.  slowly;  much  more 
freely  at  35J-38°  C.,  when  it  produces  the  coloi  more  quickly. 

RAPIDITY  OF  GROWTH. — Moderate. 

SPORE-PRODUCTION. — Not  observed. 

NEED  OF  AIR. — Does  not  grow  under  mica.  Facultatively  anaerobic,  but 
does  not  produce  color  except  with  free  access  of  oxygen. 

GAS-PRODUCTION. — Produces  faint  foul  odor. 

BEHAVIOR  TO  GELATIN.— Liquefies  gelatin  slowly. 

COLOR-PRODUCTION. — Produces  a  bluish-green  color  which  in  old  cul- 
tures changes  almost  to  a  black.  Upon  the  addition  of  acids  (both  vegetable 
and  mineral)  to  cultures  the  color  changes  to  red,  and  upon  the  addition  of 
alkalies  a  bright  grass-green  appears.  This  reaction  is  best  seen  in  bouillon 
and  gelatin  cultures,  but  occurs  in  other  media  as  well,  notably  blood-serum. 

BEHAVIOR  TO  ANILINE  DYES.— Stains  easily  and  well  with  any  of  the 
aniline  dyes  usually  employed,  and  by  Gram's  method. 

MICROSCOPIC  APPEARANCE  IN  DIFFERENT  MEDIA. — Under  the  micro- 
scope, its  general  appearance  on  various  media  is  of  a  rod  larger  than  the 
Bacillus  pyocyaneus.  In  peptone  cultures  this  difference  is  verv  marked. 
In  this  case,  the  Bacillus  pyocyaneus  tested  appeared  as  very  short,  oval, 
bacilli,  almost  like  micrococci,  while  the  new  bacillus  showed  as  a  long, 
fine  rod,  from  four  to  six  times  as  long  as  broad — length  about  one-half  the 
diameter  of  a  red-blood  corpuscle — and  arranged  sometimes  two  or  three 
end  to  end.  These  same  cultures  transferred  to  gelatin  became  indistin- 
guishable from  each  other  in  size. 

PATHOGENESIS.— Injections  of  small  quantities  (0.5  centimetre)  of  a  bouil- 
lon culture  twenty-four  hours  old  into  the  abdominal  cavity  of  rabbits  and 
guinea-pigs,  killed  fifty  per  cent  in  from  twenty-four  to  thirty-six  hours. 
Autopsy  showed  general  congestion  of  abdominal  viscera,  slight  effusion  into 
the  peritoneal  cavity,  and  cover-glass  preparations  and  cultures  showed  the 
bacilli  in  the  effusion  in  the  abdominal  cavity,  as  well  as  in  the  blood  from 
the  heart  and  various  organs. 

96.   BACILLUS  OP  FIOCCA. 

Found  by  Fiocca  in  the  saliva  of  cats  and  dogs. 

Closely  resembles  the  influenza  bacillus  of  Preiffer  and  of  Canon. 

Morphology. — Resembles  the  bacillus  of  rabbit  septicaemia,  but  is  only 
half  as  large— from  0.2  to  0.33  n  in  breadth.  The  length  is  but  little  greater 
than  the  breadth.  Usually  seen  in  pairs,  closely  resembling  diplococci. 
When  cultivated  on  potato  it  appears  to  be  a  micrococcus,  but  in  the  blood 
of  infected  animals  and  in  bouillon  cultures  it  is  seen  to  be  a  short  bacillus. 

Stains  with  difficulty  with  the  usual  aniline  colors,  but  is  readily  stained 
by  Ehrlich's  method  or  with  Ziehl's  solution. 

Biological  Characters.  — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  best 


NOT  DESCRIBED   IN   PREVIOUS   SECTIONS.  485 

at  37°  C.  and  does  not  develop  at  temperatures  below  15°  C.  In  agar  plates, 
at  37°  C.,  small,  punctiform  colonies  are  developed  at  the  end  of  twenty-four 
hours;  these  do  not  increase  in  size  later;  under  the  microscope  the  deep 
colonies  are  seen  to  be  spherical,  granular,  and  dark  yellow  in  color ;  the 
superficial  colonies  are  more  or  less  round,  with  irregular  outlines,  trans- 
parent, slightly  granular,  and  often  have  a  shining  nucleus  at  the  centre. 
Upon  gelatin  plates  the  colonies  have  a  similar  appearance,  but  are  not  vis- 
ible in  less  than  four  or  five  days.  In  streak  cultures  upon  the  surface  of 
agar  small,  punctiform  colonies  are  seen  along  the  track  of  the  needle  at  the 
end  of  twenty-four  hours,  resembling  fine  dewdrops;  the  following  day 
these  colonies  are  a  little  larger  and  less  transparent;  they  remain  distinct, 
especially  along  the  margins  of  the  line  of  growth.  Upon  potato  a  very 
thin,  transparent  layer  is  developed,  which  does  not  change  the  appearance 
of  the  surface  of  the  potato,  but  slightly  increases  its  resistance  to  the  plati- 
num needle.  In  bouillon  small  flocculi.  suspended  in  the  clear  liquid,  are 
developed  within  twenty -four  hours;  these  subsequently  sink  to  the  bottom. 

Milk  is  not  coagulated  by  this  bacillus,  and  no  gas  is  produced  in  media 
containing  sugar. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  young  rats,  and  mice, 
in  which  animals  it  produces  general  infection,  and  death-  in  rabbits— at 
the  end  of  twenty-four  hours.  The  bacillus  is  found  in  the  blood  in  great 
numbers. 

97.    PROTEUS  VULGARIS. 

Obtained  by  Hauser  (1885)  from  putrefying  animal  substances, 
and  since  shown  to  be  one  of  the  most  common  and  widely  distrib- 
uted putrefactive  bacteria.  This  and  the  other  species  of  Proteus 


FIG.  156.— Proteus  vulgaris;  '*  swarming  islands  "  from  a  gelatin  culture.    X  285.    (Hauser.) 

described  by  the  same  bacteriologist  (Proteus  mirabilis,  Proteus  Zen- 
keri)  have  no  doubt  frequently  been  encountered  by  previous  observ- 
ers, and  are  among  the  species  formerly  included  under  the  name 
66  Bacterium  termo,"  which  was  applied  to  any  minute  motile  bacilli 
found  in  putrefying  infusions. 

Morphology. — Bacilli  with  rounded  ends,  about  0.6  //  broad,  and 


486  PATHOGENIC   AEROBIC   BACILLI 

varying  greatly  in  length,  being  sometimes  short  oval,  and  at  others 
from  1.25  to  3.75  /*  in  length  ;  also  grow  out  into  flexible  filaments, 
which  may  be  more  or  less  wavy  or  spiral  in  form.  The  short  rods 
are  commonly  seen  in  pairs  ;  they  have  terminal  flagella ;  involution 
forms  are  frequently  seen,  the  most  common  being  spherical  bodies 
about  1.6  //in  diameter.  In  old  cultures  in  bouillon,  or  in  cultures 
made  in  meat  infusion  in  the  incubating  oven,  the  short  oval  forms 
greatly  predominate,  but  in  recent  cultures  in  nutrient  gelatin  fila- 
ments of  considerable  length  are  encountered  in  association  with 
shorter  rods. 

Stains  readily  with  fuchsin  or  gentian  violet — not  so  well  with 
the  brown  aniline  colors  ;  does  not  stain  by  Gram's  method  (Cheyne). 

Biological  Characters. — Anaerobic  and.  facultative  anaerobic, 
liquefying,  motile  bacillus.  Grows  rapidly  in  the  usual  culture 
media  at  the  room  temperature. 

The  growth  upon  gelatin  plates  (five  per  cent  of  gelatin)  at  the 
room  temperature  is  very  characteristic  ;  at  the  end  of  six  or  eight 
hours  small  depressions  in  the  gelatin  are  observed,  which  contain 
liquefied  gelatin  and  grayish- white  masses  of  bacilli.  Under  a  low 
power  these  depressions  are  seen  to  be  surrounded  by  a  marginal 
zone  consisting  of  two  or  three  layers,  outside  of  which  is  a  zone  of  a 
single  layer,  from  which  amoeba-like  processes  extend  upon  the  sur- 
face of  the  gelatin.  These  processes  are  constantly  undergoing 
changes  in  their  form  and  position,  and  may  become  separated  from 
the  mother  colony,  or  remain  temporarily  attached  to  it  by  a  narrow 
thread  consisting  of  bacilli ;  after  a  time  the  entire  surface  of  the 
gelatin  is  covered  with  wandering,  amoeba-like  colonies ;  these 
rapidly  cause  liquefaction,  which  by  the  end  of  twenty-four  to  forty- 
eight  hours  has  reached  a  depth  of  one  millimetre  or  more  over  the 
entire  surface.  The  deep  colonies  also  are  surrounded  by  processes 
projecting  into  the  gelatin,  which  may  be  observed  to  suddenly  ad- 
vance and  again  to  be  retracted  towards  the  central  zoogloea-like 
mass.  Liquefaction  around  the  colony  rapidly  progresses,  and 
actively  motile  rods  and  spiral  filaments  may  be  seen  about  the  peri- 
phery of  this  liquefied  gelatin,  while  about  it  is  a  radiating  crown  of 
irregular  processes,  some  of  which  may  be  screw-like  or  corkscrew- 
formed.  In  ten-per-cent  gelatin  the  migration  of  surface  colonies, 
above  described,  is  not  observed.  In  gelatin  stick  cultures  liquefac- 
tion occurs  along  the  entire  line  of  puncture,  and  soon  the  contents 
of  the  tube  are  completely  liquefied  ;  near  the  surface  of  the  liquefied 
gelatin  the  growing  bacilli  form  a  grayish-white  cloudiness,  and  at 
the  bottom  of  the  tube  an  abundant  flocculent  deposit  is  formed. 
Upon  the  surface  of  nutrient  agar  a  rapidly  extending,  moist,  thin, 
grayish- white  layer  is  formed.  Upon  potato  this  bacillus  produces  a 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  487 

dirty-white,  moist  layer.  The  cultures  in  media  containing  albumin 
or  gelatin  have  a  putrefactive  odor  and  acquire  a  strongly  alkaline 
reaction.  A  temperature  of  20°  to  24°  C.  is  most  favorable  for  the 
growth  of  this  bacillus.  It  is  a  facultative  anaerobic  and  grows  in 
an  atmosphere  of  hydrogen  or  of  carbon  dioxide,  although  not  so 
rapidly  as  in  the  presence  of  oxygen.  The  movements  are  often  ex- 
tremely active  and  difficult  to  follow  under  the  microscope  ;  again 
they  may  be  quite  deliberate,  or  the  bacilli  may  remain  motionless 
for  a  time  and  again  dart  off  in  active  motion.  The  long  terminal 
flagella  may  sometimes  be  discerned  by  means  of  a  good  objective 
and  careful  manipulation  of  the  light. 

Pathogenesis. — Pathogenic  for  rabbits  and  for  guinea-pigs  when 
injected  into  the  circulation,  into  the  cavity  of  the  abdomen,  or  sub- 
cutaneously  in  considerable  quantity.  Cultures  in  nutrient  gelatin 
are  said  by  Cheyne  to  be  more  pathogenic  (toxic)  than  those  in  bouil- 
lon. When  injected  into  the  muscles  of  rabbits  a  much  smaller 
dose  produces  a  fatal  result  than  when  injected  subcutaneously. 
In  Cheyne's  experiments,  made  in  London  (1886),  one-tenth  cubic 
centimetre  of  a  liquefied  gelatin  culture,  injected  into  the  dorsal 
muscles,  was  invariably  fatal  in  from  twenty-four  to  thirty-six  hours; 
a  dose  of  one-twentieth  cubic  centimetre,  injected  in  the  same  way, 
usually  caused  death;  while  one- fortieth  cubic  centimetre  gave  rise  to 
an  extensive  local  abscess,  and  the  animals  died  at  the  end  of  six  or 
eight  weeks.  Doses  of  less  than  one-five-hundredth  cubic  centimetre 
produced  no  effect.  Cheyne  estimates  that  one  cubic  centimetre  of  a 
culture  in  nutrient  gelatin  contains  4,500,000,000  bacilli,  and,  conse- 
quently, that  a  smaller  number  than  9,000,000  produced  no  effect  when 
injected  into  the  muscular  tissue  of  rabbits.  Injections  into  the  sub- 
cutaneous connective  tissues  of  a  dose  twice  as  large  as  that  which  in- 
variably proved  fatal  when  injected  into  the  muscles  usually  caused 
an  extensive  abscess,  but  did  not  kill  the  animal;  and,  after  re- 
covery from  the  effects  of  such  an  injection,  the  rabbit  was  found  to 
be  immune  against  a  similar  dose  injected  into  the  muscles.  Foa 
and  Bonome  have  succeeded  in  producing  immunity  against  the 
effects  of  virulent  cultures  of  this  bacillus  by  inoculating  rabbits  with 
filtered  cultures,  and  also  by  injecting  beneath  the  skin  of  these  ani- 
mals a  solution  of  neurin,  which  they  believe  to  be  the  principal 
toxic  product  present  in  the  cultures. 

Proteus  Vulgar  is  in  Cholera  Infantum. — The  extended  re- 
searches of  Booker  have  led  him  to  the  conclusion  that  this  bacillus 
plays  an  important  part  in  the  production  of  the  morbid  symptoms 
which  characterize  cholera  infantum.  Proteus  vulgaris  was  found 
in  the  alvine  discharges  in  a  considerable  proportion  of  the  cases  ex- 
amined, but  was  not  found  in  the  faeces  of  healthy  infants.  "  The 


488  PATHOGENIC   AEROBIC  BACILLI 

prominent  symptoms  in  the  cases  of  cholera  infantum  in  which  the 
proteus  bacteria  were  found  were  drowsiness,  stupor,  emaciation 
and  great  reduction  in  flesh,  more  or  less  collapse,  frequent  vomiting 
and  purging,  with  watery  and  generally  offensive  stools." 

The  researches  of  Krogius,  Schnitzler,  Schmidt  and  Aschoff,  and 
others,  show  that  in  cases  of  c}'stitis  and  of  pyelonephritis  this  bacil- 
lus is  frequently  found  in  pure  cultures,  or  associated  with  other  bac- 
teria. The  authors  last  named  state  that  in  sixty  cases  of  cystitis 
reported  by  various  authors  the  colon  bacillus  was  found  in  pure  cul- 
tures, and  in  thirteen  cases  the  proteus  of  Hauser.  Next  to  Bacillus 
coli  communis  Proteus  vulgar  is  appears  to  be  the  microorganism 
most  frequently  concerned  in  the  etiology  of  pyelonephritis. 

Levy  (1895)  isolated  from  sour  yeast  a  bacillus,  which  he  identified 
as  "Proteus  Hauseri,"  and  made  numerous  experiments  on  dogs  to 
test  its  pathogenic  power.  From  five  to  ten  cubic  centimetres  of  a 
liquefied  gelatin  culture  injected  into  the  circulation,  through  a  vein, 
caused  the  typical  symptoms  of  "sepsin  poisoning,"  as  formerly  de- 
scribed by  Bergmann  and  Schmeideberg  (1868).  In  two  dogs  which 
died  at  the  end  of  forty-eight  hours  the  intestinal  tract  was  found  in 
a  condition  of  intense  hemorrhagic  infiltration.  The  spleen  and 
glands  of  the  mesentery  were  much  enlarged.  But  a  bacteriological 
examination  gave  an  entirely  negative  result,  showing  that  death 
resulted  from  toxemia  and  not  from  septicaemia.  Further  experi- 
ments showed  that  the  dried  precipitate  obtained  from  liquefied  gela- 
tin cultures,  by  the  addition  of  alcohol,  had  the  same  pathogenic 
action  on  dogs,  rabbits,  and  mice  as  cultures  containing  the  living 
bacilli.  That  a  similar  pathogenic  effect  is  produced  in  man  by  the 
products  of  growth  of  this  bacillus  was  shown  by  the  following  facts: 
While  conducting  his  experiments  Levy  had  an  opportunity  to  make 
a  bacteriological  examination  in  the  case  of  a  man  who  died  after  a 
brief  attack  of  cholera  morbus.  From  the  vomited  material  and  the 
stools  he  obtained  a  pure  culture  of  proteus ;  but  the  blood,  collected 
at  the  autopsy,  was  sterile.  In  the  mean  time  seventeen  other  per- 
sons who  had  eaten  at  the  same  restaurant  were  taken  sick  in  the  same 
way.  Upon  an  examination  at  the  restaurant  it  was  found  that  the 
bottom  of  the  ice  chest  in  which  the  proprietor  kept  his  meats  was 
covered  with  a  slimy,  brown  layer,  which  gave  off  a  disagreeable 
odor.  Cultures  from  this  gave  the  proteus  as  the  principal  micro- 
organism present.  Levy  concludes  from  his  own  investigations  and 
those  of  other  bacteriologists  that  in  so-called  "  flesh-poisoning  "  bac- 
teria of  this  group  are  chiefly  at  fault,  and  that  the  pathogenic  effects 
are  due  to  toxic  products  evolved  during  their  development. 


NOT  DESCRIBED   IN  PREVIOUS   SECTIONS.  489 

98.    PROTEUS  OF  KARLINSKI. 

Synonym. — Bacillus  murisepticus  pleomorphus  (Karlinski).  Probably 
identical  with  Proteus  vulgaris  of  Hauser. 

Obtained  by  Karlinski  (1889)  from  a  fibro-purulent  uterine  discharge,  and 
from  abscesses  in  the  uterus  and  its  appendages  in  a  puerperal  woman. 

Morphology.  —Resembles  Proteus  vulgaris  of  Hauser  in  its  morphology, 
and  presents  various  forms  under  different  circumstances  relating  to  the 
culture  medium,  the  temperature,  age  of  culture,  etc. — sometimes  as  spheri 
cal  or  short  oval  cells,  at  others  as  longer  or  shorter  rods  or  spiral  filaments; 
usually  as  bacilli  with  round  ends  two  and  a  half  times  as  long  as  thick, 
often  united  in  pairs. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  arid  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Spore  formation  not  observed.  Grows  rapidly  in  the 
usual  culture  media  at  the  room  temperature.  In  gelatin  plate  cultures,  at 
the  end  of  ten  hours,  small  colonies  are  developed  which  nave  well-defined 
outlines,  are  oval  or  whetstone-shaped,  of  a  light-brown  color  by  transmitted 
light  and  white  by  reflected  light,  with  a  somewhat  darker  margin  and  a 
smooth  surface,  sometimes  marked  by  shallow  clef ts ;  at  the  end' of  twenty 
hours  the  colonies  commence  to  have  irregular  margins,  and  the  surface  of 
the  gelatin  above  them  is  marked  by  concentric  rings.  At  the  end  of  thirty 
hours  the  colonies  have  formed  a  bulb-shaped  liquefaction  of  the  gelatin, 
and  delicate,  ray  like  offshoots  are  seen  around  the  margin.  At  the  end  of 
two  days  the  bulbous  cavities  are  about  one  and  a  half  millimetres  in  diameter 
and  contain  a  cloudy,  grayish -white  liquid ;  they  are  surrounded  by  a  moist- 
looking,  gray,  irregular  marginal  zone.  In  gelatin  stick  cultures,  at  the  end 
of  twenty-four  hours,  a  funnel  shaped  liquefaction  of  the  gelatin  occurs  near 
the  surface,  and  a  grayish- white,  cloudy  mass  is  developed  along  the  line  of 
puncture;  at  the  end  of  forty- eight  hours  a  sac-like  pouch  of  liquefied  gela- 
tin has  formed,  and  in  the  course  of  four  or  five  days  the  gelatin  is  entirely 
liquefied.  Upon  agar  plates  the  colonies  are  at  first  oval  in  form  and  white 
by  reflected  light,  or  pale  brown  by  transmitted  light  ;  at  the  end  of  thirty 
hours  the  surface  becomes  wrinkled  or  folded  and  is  surrounded  by  radiat- 
ing, delicately  twisted  offshoots.  Upon  the  surface  of  agar  a  white  layer 
is  developed.  Upon  potato  a  whitish-gray,  soft,  homogeneous  layer,  which 
after  standing  along  time  has  a  darker  'color.  Upon  blood  serum  a  thin, 
grayish-white  layer  is  formed  and  the  serum  is  rapidly  liquefied.  Gelatin 
cultures  acquire  a  strongly  alkaline  reaction  and  give  off  a  disagreeable 
odor  resembling  that  of  butyric  acid. 

Pathogenesis. — White  mice  inoculated  at  the  root  of  the  tail  die  in  from 
twenty-two  to  twenty-four  hours  ;  the  spleen  is  greatly  enlarged;  the  bacilli 
are  found  in  blood  from  the  various  organs — less  numerous  in  blood  from 
the  heart.  Field  mice  and  house  mice  are  less  susceptible.  Subcutaneous 
injections  in  rabbits  may  give  rise  to  local  inflammation  and  also  to  general 
infection.  In  white  rats  and  guinea-pigs  a  local  abscess  may  result  from  a 
subcutaneous  inoculation, 

99.    PROTEUS  MIRABILIS. 

Obtained  by  Hauser  (1885)  from  putrefying  animal  substances. 

Morphology. — Bacilli  resembling  very  closely  the  preceding  species  (Pro- 
teus vulgaris),  but  presenting  more  numerous  involution  forms,  which  may 
be  spherical,  pear-shaped,  or  spermatozoa-like,  etc.  The  bacilli  are  about 
0  6  //  in  diameter  and  vary  greatly  in  length,  being  sometimes  nearly  spheri- 
cal, or  forming  rods  of  2  to  3.75  //  in  length,  or  long  filaments. 

Biological  Characters. — An  aerobic  said  facultative  anaerobic,  liquefy- 
ing,  motile  bacillus.  Spore  formation  has  not  been  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Does  not  liquefy  gelatin  as 


490 


PATHOGENIC  AEROBIC  BACILLI 


rapidly  as  Proteus  vulgaris.  Upon  gelatin  plates,  at  the  end  of  twelve 
hours,  superficial  colonies  of  two  to  three  millimetres  in  diameter  are  formed ; 
under  a  low  power  these  appear  finely  granular  and  brownish  in  color,  and 
have  an  irregular  outline;  outgrowths  from  the  margin  extend  in  various 
directions  and  form  new  colonies,  which  may  be  attached  for  a  time  by  a 
long  and  slender  thread  consisting  of  bacilli.  The  movement  of  these  new 
colonies  is  not  as  pronounced  as  in  the  case  of  the  preceding  species,  and 


Fio.  157.—"  Swarming  islands  "  of  Proteus  mirabilis,  from  a  gelatin  culture,    x  285.    (Hauser.) 

they  are  characterized  by  the  presence  of  numerous  distorted  bacilli — invo- 
lution forms.    The  deep  colonies  form  spiral  zoogloea  masses. 

In  gelatin  stick  cultures  the  whole  surface  is  first  covered  with  threads 
and  islands  of  bacilli,  which  after  a  time  form  an  anastomosing  network,  and 
finally  a  confluent  layer  which  at  the  end  of  forty-eight  hours  is  rather  thick, 


FIG.  158.— Spiral  zooglosa  from  a  culture  of  Proteus  mirabilis.    X  95.    (.Hauser.) 

with  a  moist,  shining  surface  and  grayish  color,  and  appears  to  be  perforated 
with  numerous  small,  sieve-like  openings.  These  thinner  and  transparent 
places  disappear  after  a  time,  and  at  the  end  of  two  or  three  days  liquefac- 
tion of  the  gelatin  commences;  complete  liquefaction  does  not  occur  until 
the  fifth  or  sixth  day,  or  even  later.  Along  the  line  of  puncture  finely  gran- 
ular colonies  are  first  formed,  from  which  long  threads  are  given  off,  which 
form  after  a  short  time  a  tolerably  broad  zone  of  threads  and  spiral  zoogloea 
masses. 


NOT  DESCRIBED   IN  PREVIOUS  SECTIONS.  491 

Pathogenesis. — In  Hauser's  experiments  filtered  cultures  (two  to  six  cubic 
centimetres),  injected  into  the  circulation  or  into  the  cavity  of  the  abdomen 
in  rabbits,  caused  fatal  toxaemia. 

100.    PROTEUS  ZENKERI. 

Obtained  by  Hauser  (1885)  from  putrefying  animal  substances. 

Morphology. — Bacilli  which  vary  greatly  in  length — average  about  1. 65 //, 
and  about  0.4  ju  broad. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Spore  formation  not  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Upon  the  surface  of  nutrient 
gelatin  a  laminated  mass  forms  about  the  point  of  puncture,  from  the  peri- 
phery of  which  offshoots  are  given  off,  at  the  extremities  of  which  colonies 
are  formed,  as  in  the  case  of  Proteus  mirabilis.  Gradually  a  rather  thick, 
grayish-white,  opaque  layer  is  formed,  which  covers  the  entire  surface  of  the 
gelatin  and  is  easily  detached  from  it.  This  species  is  distinguished  from 
the  two  preceding  by  the  fact  that  it  does  not  liquefy  gelatin  or  blood  serum 
and  does  not  give  off  a  decided  putrefactive  odor  when  cultivated  in  these 
media. 

Pathogenesis. — Considerable  quantities  injected  into  small  fl.tmna.1a  give 
rise  to  local  abscesses  and  to  symptoms  of  toxaemia. 

101.  PROTEUS   SEPTICUS. 

Obtained  by  Babes  (1889)  from  the  mucous  membrane  of  the  intestine  and 
the  various  organs  of  a  boy  who  died  of  septicaemia. 

Morphology. — Bacilli  about  0  4/z  broad  and  varying  greatly  in  length; 
slightlv  curved  rods  or  flexible  filaments,  often  associated  in  loose  chains. 

Stains  by  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacillus.  Spore 
formation  not  observed.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  In  gelatin  plates  centres  of  liquefaction  are  quickly  formed 
and  rapidly  extend.  The  spherical,  liquefied  places  have  at  first  a  wavy  or 
dentate  outline,  and  are  surrounded  by  a  branching,  transparent,  granular 
margin  which  rapidly  extends  in  advance  of  the  liquefaction.  In  stick  cul- 
tures in  nutrient  gelatin  liquefaction  of  the  entire  con  tents  of  the  tube  may 
take  place  within  twenty-four  hours,  or  a  broad,  liquefied  sac  is  formed 
along  the  line  of  puncture.  Gelatin  cultures  give  off  a  very  disagreeable 
odor.  Upon  the  surface  of  nutrient  agar,  at  37°  C.,  a  peculiar,  thick  net- 
work extends  over  the  surface  in  the  course  of  a  few  hours.  Upon  potato  an 
elevated,  brownish-white,  shining  layer  is  formed.  Blood  serum  is  lique- 
fied by  this  bacillus. 

Pathogenesis. — Pathogenic  for  mice,  less  so  for  rabbits.  In  mice  death 
occurs  in  from  one  to  three  days  after  the  subcutaneous  injection  of  a  small 
quantity  of  a  pure  culture  ;  the  bacilli  are  present  in  the  blood  in  small 
numbers. 

102.  PROTEUS   LETHALIS. 

Synonym. — Proteus  bei  Lungengangran  des  Menschen  (Babes). 

Obtained  by  Babes  (1889)  from  the  spleen  and  gangrenous  portions  of  the 
lung  of  a  man  who  died  of  septicaemia. 

Morphology  — Short  rods  with  round  ends,  from  0.8  to  1.5  >u  thick  ;  often 
swollen  in  the  middle,  like  a  lemon  or  a  flask  ;  forms  short,  flexible  filaments 
which  also  present  similar  swellings. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
fying,  motile  bacillus.  Not  observed  to  form  spores.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  In  gelatin  plates  forms  hemi- 
spherical, elevated,  whitish,  translucent  colonies,  which  later  send  out 


492  PATHOGENIC  AEROBIC  BACILLI 

coarse  branches  which  ramify  over  the  surface  of  the  gelatin.  A  similar 
growth  is  observed  upon  the  surface  of  gelatin  stick  cultures,  and  an  abun- 
dant development  takes  place  along  the  line  of  puncture.  Upon  nutrient 
agar  a  thick,  opaque,  slightly  yellowish  layer  is  formed.  Upon  potato  a 
moist,  shining,  brownish  layer  is  developed,  and  the  potato  acquires  a 
brownish  color.  Upon  blood  serum  the  growth  is  less  abundant  than  on 
agar;  the  blood  serum  is  not  liquefied.  This  bacillus  grows  rapidly  at  the 
room  temperature;  it  is  destroyed  by  a  temperature  of  80°  C.,  and  presum- 
ably does  not  form  spores. 

Pathogenesis. — Recent  cultures  are  very  pathogenic  for  mice  and  for 
rabbits,  less  so  for  guinea  pigs.  The  subcutaneous  injection  of  a  small 
quantity  of  a  pure  culture  kills  susceptible  animals  in  two  or  three  days. 
More  or  less  oedema  is  found  at  the  point  of  inoculation.  Injections  into  the 
rectum  of  rabbits  gave  rise  to  haemorrhagic  enteritis,  peritonitis,  and  death 
at  the  end  of  four  days. 

103.    BACILLUS  A  OF  BOOKER. 

"Obtained  by  Booker  (1889)  from  the  alvine  discharges  of  children  suffer- 
ing from  cholera  infantum. 

Morphology. — Bacilli  with  round  ends,  varying  greatly  in  length,  usually 
three  to  four  /*  long  and  0  7  JJ.  broad  (in  recent  agar  cultures).  In  older  cul- 
tures the  bacilli  are  shorter  and  smaller. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  lique- 
fying, motile  bacillus.  Grows  at  the  room  temperature  in  the  usual  culture 
media.  In  gelatin  plates  colonies  are  visible  at  the  end  of  twenty-four 
hours;  under  the  microscope  these  are  nearly  colorless,  and  liquefaction 
soon  occurs  around  them.  In  gelatin  stick  cultures  complete  liquefaction 
occurs  in  three  or  four  days.  Upon  agar  a  colorless  layer  covering  the  entire 
surface  is  developed  in  tnree  or  four  days,  and  an  abundant  development 
occurs  along  the  line  of  puncture.  Agar  colonies  have  a  bluish  look,  and 
are  surrounded  by  an  indistinct  halo  which  shades  off  gradually  into  the 
surrounding  agar  ;  under  a  low  power  the  colonies  are  light-brown  and  the 
borders  indistinct;  the  surface  has  a  delicate,  wavy  appearance.  Upon  po- 
tato the  growth  is  luxuriant  and  of  a  dirty-brown  color.  Blood  serum  is 
liquefied  by  this  bacillus. 

Milk  is  coagulated  into  a  gelatinous  mass  having  an  alkaline  reaction ; 
later  the  coagulum  is  dissolved. 

Pathogenesis. — Mice  and  guinea-pigs  fed  with  cultures  in  milk  die  in  from 
one  to  eight  days. 

104.    BACILLUS   ENDOCARDITIDIS  GRISEUS. 

Obtained  by  Weichselbaum  (1888)  from  the  affected  valves  in  a  case  of 
endocarditis  recurrens  ulcerosa. 

Morphology. — Short  rods  with  rounded  or  somewhat  pointed  ends,  about 
two  to  three  times  as  long  as  broad— of  about  the  same  dimensions  as  the 
bacillus  of  typhoid  fever." 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method;  the 
longer  rods  from  old  cultures  are  irregularly  stained. 

Biological  Characters. — An  aerobic,  non  liquefy  ing,  motile  bacillus. 
Refractive  bodies  may  be  seen  in  some  of  the  rods,  which  resemble  spores  and 
are  stained  by  the  method  of  Ernst,  but  they  do  not  show  the  resistance  of 
known  spores  to  physical  and  chemical  agents.  Grows  well  in  the  usual 
culture  media  at  trie  room  temperature.  Upon  gelatin  plates  colonies  are 
formed  which  resemble  those  of  Friedlander's  bacillus,  but  which  gradually 
acquire  a  gray  or  grayish-white  color.  The  prominent,  convex,  superficial 
colonies  under  a  low  power  are  finely  granular  and  grayish  brown  in  color; 
the  deep  colonies  are  yellowish-brown  in  color,  have  slightly  notched  mar- 
gins, and  the  surface  is  covered  with  minute  projections.  In  stick  cultures 


NOT  DESCRIBED  IN  PREVIOUS  SECTIONS.  493 

a  rather  thin,  circular  layer  forms  about  the  point  of  puncture;  this  has  the 
appearance  of  stearin ;  later  it  becomes  grayish- white  and  the  margins  are 
marked  by  radiating  lines.  Upon  the  surface  of  nutrient  agar  a  similar 
growth  occurs  which  has  a  pale-brown  or  reddish-gray  color.  Upon  potato 
in  the  incubating  oven  an  abundant  development  occurs,  forming  a  dry 
looking  layer  of  a  grayish-brown  color  and  having  irregularly  notched  mar- 
gins. Upon  blood  serum  an  abundant,  grayish-white  growth  of  cream-like 
consistence  forms  along  the  impfstrich;  later  this  has  a  reddish  gray  color. 
This  bacillus  grows  to  the  bottom  of  the  line  of  puncture  in  stick  cultures, 
and  is  no  doubt  a  facultative  anaerobic. 

Pathogenesis. — Pathogenic  for  white  mice  and  for  guinea-pigs. 

105.    BACILLUS   ENDOCARDITIDIS   CAPSULATUS. 

Obtained  by  Weichselbaum  (1888)  from  thrombi  and  embolic  infarctions 
in  the  spleen  and  kidneys  of  a  man  who  died  from  endocarditis  with  forma- 
tion of  thrombi. 

Morphology. — Resembles  Friedlander's  bacillus,  and  is  frequently  sur- 
rounded by  a  capsule,  which  may  be  stained ;  also  forms  long,  curved  fila- 
ments, in  the  protoplasm  of  which  vacuoles  may  be  observed  in  stained  pre- 
parations. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method;  by 
staining  with  fuchsin  and  carefully  decolorizing  with  diluted  alcohol  the 
presence  of  a  capsule  may  be  demonstrated. 

Biological  Characters.—  An  aerobic,  non -liquefying  bacillus.  Grows  in 
the  usual  culture  media  at  the  room  temperature. 

In  gelatin  stick  cultures  development  occurs  along  the  line  of  puncture, 
and  on  the  surface  as  a  rather  thin,  white,  dry  layer  which  resembles  stearin. 
In  agar  plates  the  superficial  colonies  are  thin,  about  two  millimetres  in 
diameter  and  gray  in  color ;  under  a  low  power  the  margins  are  trans- 
parent and  colorless,  and  the  centre  resembles  the  deep  colonies;  these  are 
very  small  and  grayish  white  in  color  ;  under  a  low  power  the  surface  is 
seen  to  be  covered  with  tooth-like,  projecting  masses,  the  margin  is  dentate 
and  has  a  pale-yellow  color,  while  the  centre  is  yellowish-brown. 

Pathogenesis. — Rabbits  are  killed  by  the  injection  of  a  considerable  quan- 
tity of  a  pure  culture  into  the  cavity  of  the  abdomen  or  subcutaneously. 

106.    BACILLUS   OF  LESAGE. 

Obtained  by  Lesage  (1887)  from  the  green-colored  discharges  of  infants 
suffering  from  "  green  diarrhoea,"  and  supposed  to  be  the  cause  of  this  com- 
plaint (?).  According  to  Baumgarten,  this  bacillus  is  probably  identical 
with  a  well-known  pigment-producing  saprophyte— the  Bacillus  fluorescens 
non  liquefaciens. 

Morphology. — Small  bacilli  with  round  ends,  about  2  4  /*  long  and  0.75  to 
I/*  broad ;  in  old  cultures  may  grow  out  into  long  filaments. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters  — An  aerobic,  non-liquefying  (slight  liquefaction 
in  old  cultures),  motile  bacillus.  Forms  spores.  Grows  slowly  at  the  room 
temperature  in  the  usual  culture  media,  more  rapidly  at  25°  to  35°  C.  Upon 
gelatin  plates  superficial  colonies  are  formed  which  have  irregularly  dentate, 
leaf-like  margins  and  a  smooth  surface ;  they  produce  a  greenish  color  in  the 
gelatin.  In  gelatin  stick  cultures  a  thin,  smooth,  transparent,  greenish 
layer  forms  upon  the  surface,  and  in  the  course  of  four  or  five  days  the  gela- 
tin has  acquired  throughout  a  bright-green  color.  Upon  potato  a  dark- 
green  layer  is  formed.  The  cultures  have  the  odor  of  old  urine. 

Pathogenesis. — The  injection  of  a  considerable  quantity  of  a  pure  culture 
into  the  ear  vein  of  a  rabbit  is  said  to  have  produced  green  diarrhoea,  and 
the  same  result  was  obtained  by  mixing  cultures  with  the  food  of  these  ani- 
mals. These  results  have  not  yet  been  confirmed  by  other  investigators. 


494  PATHOGENIC   AEROBIC   BACILLI 

107.    BACILLUS  OF  DEMME. 

Obtained  by  Demme  (1888)  from  the  fluid  contents  of  the  tumors  and 
pustules  of  erythema  nodosum,  and  also  from  the  blood  of  the  affected  indi- 
vidual. 

Morphology.—  Bacilli  with  round  ends,  from  2.2  to  2.5  //  long  and  0.5  to 
0.7/u  broad;  usually  collected  in  smaller  or  larger  groups. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic  (facultative  anaerobic?)  bacillus, 
which  does  not  grow  in  nutrient  gelatin  at  the  room  temperature.  Grows 
in  nutrient  agar  at  35°  to  37°  C.  Forms  spores.  In  agar plates,  at  35°  to  37° 
C.,  smooth,  spherical,  shining  white  colonies  are  formed  in  from  forty-eight 
to  sixty  hours,  whicli  at  the  end  of  six  or  seven  days  may  have  the  size  of  a 
small  coin — five  centimes;  these  are  marked  by  lines  radiating  from  the 
centre,  which  are  slightly  elevated  above  the  surface  of  the  colony  and  have 
a  silvery  lustre  by  obliquely  reflected  light;  the  margins  of  the  colony  are 
fringe-like,  and  after  ten  or  twelve  days  conical  offshoots  are  given  off  from 
this  thready  margin.  In  agar  stick  cultures  growth  occurs  along  the  line 
of  puncture  in  the  form  of  a  thorny  column  which  has  a  paraffin-like 
lustre. 

Pathogenesis. — According  to  Demme,  when  injected  subcutaneously  into 
guinea-pigs,  or  by  rubbing  pure  cultures  into  the  scarified  skin,  an  eruption 
occurs  which  resembles  that  of  erythema  nodosum  and  is  followed  by  a 
gangrenous  condition  of  the  skin.  Rabbits,  dogs,  and  goats  proved  to  be 
refractory. 

108.    BACILLUS  CEDEMATIS  AEROBICUS. 

Synonym. — A  new  bacillus  of  malignant  osdema  (Klein). 

Obtained  from  garden  earth  by  inoculation  in  guinea-pigs. 

Morphology. — Bacilli  from  0.8  to  2.4  //  in  length  and  0.7  u  thick;  grow 
out  into  long  filaments. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  diameters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, motile  bacillus.  Does  not  form  spores.  Grows  at  the  room  tempera- 
ture in  the  usual  culture  media.  Upon  gelatin  plates,  at  the  end  of  twenty- 
four  hours,  small,  gray,  punctiform  colonies  are  developed ;  at  the  end  of 
forty-eight  hours  the  superficial  colonies  are  seen  as  flat,  grayish,  transparent 
plaques,  the  margins  of  which  are  thin  and  irregularly  notched ;  these  attain 
a  diameter  of  several  millimetres  in  the  course  of  a  few  days.  The  deep  colo- 
nies do  not  exceed  the  diameter  of  a  pin's  head ;  they  remain  spherical,  and 
by  transmitted  light  have  a  brownish  color.  In  gelatin  stick  cultures  a, 
white  line  of  growth  is  developed  along  the  track  of  the  inoculating  needle, 
and  at  the  bottom  of  this  isolated,  punctiform  colonies  are  seen ;  upon  the 
surface  a  flat,  thin,  transparent,  grayish  layer  with  a  dentate  margin  is 
developed.  Upon  the  surface  of  agar  a  smeary,  grayish-white  stripe  is  de- 
veloped along  the  impfstrich.  Alkaline  bouillon,  at  the  end  of  twenty-four 
hours  at  37J  C.,  is  densely  clouded,  and  later  contains  numerous  flocculi,  but 
no  pellicle  upon  the  surface ;  at  the  end  of  twenty-four  hours  the  reaction 
becomes  strongly  alkaline.  Upon  potato  a  viscid,  yellowish  stripe  is  devel- 
oped along  the  line  of  inoculation.  In  deep  cultures  in  nutrient  gelatin  gas 
bubbles  are  developed  in  from  twenty-four  to  forty-eight  hours;  these  are 
attached  to  the  isolated  colonies. 

Pathogenic  for  guinea-pigs,  rabbits,  and  white  mice.  The  animals  die 
within  twenty  four  hours — when  very  small  quantities  are  injected  subcu- 
taneously into  guinea-pigs  they  may  live  for  two  or  three  days  and  sometimes 
recover.  The  lethal  dose  of  a  bouillon  culture  is  from  one-fourth  to  one- 
half  cubic  centimetre,  but  one  drop  of  the  cedematous  fluid  from  the  subcu- 
taneous connective  tissue  of  an  inoculated  animal  is  infallibly  fatal.  In 
guinea-pigs  an  extensive  inflammatory  oedema  is  produced  by  subcutaneous 
inoculations ;  the  spleen  is  but  slightly  enlarged.  In  rabbits  but  slight  oedema 


NOT  DESCRIBED   IN  PREVIOUS  SECTIONS.  495 

and  a  small  spleen.  In  mice  no  oedema  and  a  slightly  enlarged  spleen.  The 
bacilli  are  found  in  the  blood  of  the  heart  in  small  numbers,  and  are  some- 
what more  numerous  in  the  spleen,  especially  in  mice. 

109.    BACILLUS   OF  LETZERICH. 

Obtained  by  Letzerich  (1887)  from  the  urine  of  children  suffering  from 
"nephritis  interstitialis  primaria."  Etiological  relation  not  satisfactorily 
demonstrated. 

Morphology. — Bacilli  with  round  ends,  straight  or  slightly  curved,  often 
forming  filaments. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters.—  An  aerobic,  liquefying  bacillus.  Forms  spores. 
Grows  rapidly  in  nutrient  gelatin  at  a  comparatively  low  temperature — best 
at  14°  C.  Upon  gelatin  plates,  at  14°  C.,  complete  liquefaction  has  occurred 
in  from  thirty-six  to  forty-eight  hours,  and  a  thin,  white  film  covers  the 
surface  of  the  liquefied  gelatin;  the  same  in  gelatin  stick  cultures. 

Pathogenesis. — Rabbits  injected  in  the  cavity  of  the  abdomen  are  said  to 
die  in  about  fourteen  days.  The  autopsy  shows  an  extensive  abscess,  en- 
largement and  congestion  of  the  kidneys,  enlarged  spleen,  etc.  The  bacilli 
are  found  in  great  numbers  in  all  of  the  organs. 

110.    BACILLUS  OP  SCHIMMELBUSCH. 

Obtained  by  Schimmelbusch  (1889)  from  the  necrotic  tissues  at  the  boun- 
dary line  of  the  still  living  tissues  in  cancrum  oris,  or  noma.  Etiological 
relation  not  proved. 

Morphology. — Small  bacilli  with  round  ends;  often  united  in  pairs; 
may  grow  out  into  long  filaments. 

Stains  best  with  an  aqueous  solution  of  gentian  violet;  does  not  stain  by 
Gram's  method. 

Biological  Characters. — An  aerobic,  non-liquefying  bacillus.  Grows  in 
the  usual  culture  media  at  the  room  temperature— better  in  the  incubating 
oven  at  30°  to  37°  C.  Upon  gelatin  plates  forms  below  the  surface  spheri- 
cal, finely  granular,  grayish-white  colonies,  which  come  to  the  surface  and 
form  elevated  masses  with  slightly  dentate  margins  and  an  irregularly  cleft 
surface.  In  gelatin  stick  cultures  the  growth  along  the  line  of  inoculation 
is  coarsely  granular ;  upon  the  surface  a  broad,  flat  layer.  Upon  the  sur- 
face of  agar,  in  twenty-four  hours  at  37°  C.,  a  grayish- white  layer  along  the 
line  of  inoculation,  which  is  smooth  and  about  three  millimetres  in  breadth. 
Upon  potato,  at  the  end  of  two  weeks,  a  broad,  moist,  grayish- white  layer 
from  two  to  three  millimetres  wide.  Upon  coagulated  ascitic  fluid,  at  the 
end  of  twenty-four  hours,  a  thin  layer  along  the  impfstrich,  from  which 
lateral  offshoots  are  given  off. 

Pathogenesis.—  Cultures  injected  subcutaneously  into  rabbits  produced 
local  abscesses  only ;  not  pathogenic  for  mice  or  pigeons. 

111.   BACILLUS  FCETIDUS  OZ^EN^E. 

Obtained  by  Hajek  (1888)  from  the  nasal  secretions  of  patients  with  ozae- 
na.  Etiological  relation  not  proved. 

Morphology. — Short  bacilli,  but  little  longer  than  broad;  usually  in  pairs, 
or  in  chains  of  six  to  ten  elements. 

Stains  with  Loffler's  solution  of  methylene  blue  or  solutions  of  aniline 
colors  in  aniline  water — not  so  well  in  aqueous  solutions;  does  not  stain  by 
Gram's  method. 

Biological  Characters.  — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Spore  formation  not  observed.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  Upon  gelatin  plates  the  colonies, 
at  the  end  of  thirty-six  hours,  are  scarcely  visible,  with  well-defined  but 


496  PATHOGENIC   AEROBIC   BACILLI 

somewhat  irregular  outlines;  later  liquefaction  commences  and  crater-like 
depressions  in  the  gelatin  are  formed,  in  which  a  gas  bubble  is  seen ;  com- 
plete liquefaction  occurs  in  the  course  of  a  few  days.  In  gelatin  stick  cul- 
tures liquefaction  occurs  all  along  the  line  of  inoculation,  and  is  complete 
at  the  end  of  from  eight  to  fourteen  days.  Upon  agar  plates  the  colonies 
are  granular  in  the  centre,  and  the  margins,  under  a  low  power,  are  seen  to 
be  fringed.  Upon  the  surface  of  agar  a  moist,  slimy  layer  is  formed  along 
the  impfstrich.  Upon,  potato,  at  the  end  of  twenty-four  hours,  a  yellowish- 
brown  layer  is  formed.  Upon  blood  serum  development  is  rapid  in  the  form 
of  a  whitish  layer,  which  extends  over  the  whole  surface.  The  cultures, 
and  especially  those  kept  in  the  incubating  oven,  give  off  a  disagreeable 
putrefactive  odor,  which  is  most  intense  in  the  blood-serum  cultures. 

Pathogenesis. — Pathogenic  for  mice.  When  injected  subcutaneously 
into  rabbits  it  gives  rise  to  intense  local  inflammation  and  progressive  gan- 
grene of  the  connective  tissue. 

112.  BACILLUS  OF  LUMNITZER. 

Obtained  by  Lumnitzer  (1888)  from  the  bronchial  secretions  of  persons 
suffering  from  *'  putrid  bronchitis."  Etiological  relation  not  demonstrated. 

Morphology. — Bacilli  with  round  ends,  from  1.5  to  2n  long,  somewhat 
curved. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  motile  bacillus.  Does  not  grow  in 
nutrient  gelatin  at  the  room  temperature.  Grows  slowly  upon  agar  and 
more  rapidly  upon  blood  serum  at  36°  to  38°  C.  Forms  spores.  Upon  agar 
plates,  at  37°  C  ,  small,  grayish-white  colonies  are  formed  in  two  or  three 
uays;  upon  the  surface  these  form  hemispherical  masses  which  slowly  in- 
crease in  size.  At  the  end  of  six  or  seven  days  the  cultures  give  off  a  dis- 
agreeable odor,  quite  like  that  given  off  by  the  sputum  of  the  cases  of  putrid 
bronchitis  from  which  the  bacillus  was  obtained.  Upon  the  surface  of 
blood  serum  the  growth  is  rapid  and  forms  grayish-white,  shining  colonies, 
of  about  one  millimetre  in  diameter,  which  become  confluent  at  the  end  of 
about  four  days,  and  cover  the  entire  surface  in  eight  or  nine  days. 

Pathogenesis. — Causes  a  purulent  inflammation  when  injected  into  the 
lungs  of  rabbits,  which  involves  the  bronchial  tubes,  the  blood  vessels,  and 
the  pulmonary  alveoli ;  when  injected  subcutaneously  produces  inflamma- 
tion and  necrosis  of  the  tissues. 

113.  BACILLUS  OF  TOMMASOLI. 

Obtained  by  Tommasoli  (1889)  from  the  hairs  of  the  head  of  a  patient  suf- 
fering from  a  form  of  sycosis  supposed  to  be  due  to  the  presence  of  this 
parasite  (?). 

Morphology.—  Short,  straight  bacilli,  with  round  ends,  from  1  to  1.8  /* 
long-  and  from  0.25  to  0.3  n  broad  ;  often  united  in  chains  containing  four 
to  six  elements. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motile  bacil- 
lus. Does  not  form  spores.  Grows  slowly  at  the  room  temperature  in  the 
usual  culture  media.  Upon  gelatin  plates,  at  the  end  of  four  days,  the  deep 
colonies  are  seen  as  small,  white  points,  the  superficial  colonies  as  smooth 
discs  of  a  grayish  color.  At  the  end  of  a  month  the  deep  colonies  may  be  as 
large  as  a  mustard  seed;  the  superficial  are  thin,  shining,  and  slimy,  and 
have  a  diameter  of  one  to  two  millimetres.  In  gelatin  stick  cultures  a  con- 
vex, shining,  white  mass  is  developed  at  the  point  of  inoculation,  and  along 
the  line  of  puncture  in  the  course  of  five  or  six  days  a  white  line  of  growth 
is  seen  which  consists  of  closely  crowded,  small  colonies.  Upon  agar  the 
development  is  very  slow,  and  forms  at  first  thin,  slimy,  grayish-white 
patches  which  are  distributed  along  the  impfstrich ;  later  these  become  con- 


T   DESCRIBED   IN   PREVIOUS   SECTIONS.  497 

fluent  and  form,  shining,  wavy  stripes.  Upon  potato  the  development  is 
more  rapid  and  forms  elevated,  sharply  denned  colonies,  of  granular  ap- 
pearance and  of  a  chamois-yellowish-white  color ;  later  these  become  conflu- 
ent; the  potato  acquires  a  dark-gray  color  and  the  culture  gives  off  an  in- 
tensely disagreeable  odor. 

Pathogenesis. — Pure  cultures  rubbed  into  the  skin  of  man  produce,  at 
the  end  of  twenty-four  hours,  intense  itching,  redness,  and  a  vesicular  erup- 
tion about  the  hairs ;  at  the  end  of  three  days  small  pustules  are  formed, 
from  which  pure  cultures  may  be  recovered  (Tommasoli) .  Subcutaneous  in- 
jection into  a  rabbit  produced  no  other  result  than  the  formation  of  a  small 
abscess. 

114.    BACILLUS  OF  SCHOU. 

Obtained  by  Schou  (1885)  in  rabbits  suffering  from  vagus  pneumonia 
resulting  from  section  of  the  vagi ;  found  also  in  the  buccal  secretions  of  a 
healthy  rabbit — one  out  of  twenty-five  examined. 

Morphology. — Described  as  elliptical  cocci,  or  diplococci,  or  as  short, 
thick  bacilli. 

Stains  with  the  aniline  colors  usually  employed,  but  not  by  Gram's 
method. 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacillus.  Grows 
in  the  usual  culture  media  at  the  room  temperature.  In  gelatin  plates 
forms  spherical,  opaque,  granular  colonies  having  a  slightly  rough  surface. 
At  the  end  of  twenty -four  hours,  under  the  microscope,  active  movements 
are  observed  in  these  colonies,  which  are  surrounded  by  a  zone  of  diverging 
rays.  In  gelatin  stick  cultures  liquefaction  quickly  occurs,  and  a  copious 
white  deposit,  consisting  of  bacilli,  is  seen  at  the  bottom  of  the  tube. 

Pathogenesis. — Pure  cultures  injected  into  the  trachea,  the  pleural 
cavity,  or  the  lungs  are  said  to  have  produced  fatal  pneumonia  in  rabbits;  a 
similar  result  was  obtained  from  inhalation  experiments. 

115.    BACILLUS  NECROPHORUS. 

Obtained  by  Lofner  (1884)  from  rabbits  which  had  been  inoculated  in  the 
anterior  chamber  of  the  eye  with  small  fragments  of  a  broad  condyloma. 

Morphology. — Bacilli  of  various  lengths,  often  forming  long,  slender, 
wavy  filaments. 

Biological  Characters. — Does  not  grow  in  the  ordinary  culture  media, 
but  may  be  cultivated  in  neutral  rabbit  bouillon ;  a  less  favorable  medium  is 
blood  serum  from  the  horse.  When  small  fragments  of  the  organs  of  an 
infected  animal  are  placed  in  rabbit  bouillon  they  become  enveloped,  in  the 
course  of  three  or  four  days,  in  a  cotton-like  mass  of  filaments ;  later  white 
flocculi  are  distributed  tlirough  the  medium,  which  consist  of  similar  fila- 
ments loosely  interlaced.  The  filaments  may  present  swellings  here  and 
there,  which  are  supposed  to  represent  involution  forms. 

Pathogenesis. — Rabbits  inoculated  in  the  ear  or  in  the  anterior  chamber 
of  the  eye  with  the  flocculi  from  a  bouillon  culture,  or  with  a  small  frag- 
ment of  one  of  the  organs  of  an  infected  animal,  usually  die  at  the  end  of 
eight  days.  At  the  autopsy  a  necrotic,  cheesy  process  is  found  at  the  point 
of  inoculation,  and  purulent  foci,  surrounded  by  inflamed  or  necrotic  areas, 
in  the  lungs ;  also  purulent  collections  in  the  myocardium ;  these  were  the 
principal  pathological  changes,  but  sometimes  nodules  were  found  in  the 
abdominal  viscera.  The  slender  bacilli  described  were  found  in  all  of  these 
localized  centres  of  infection.  Pathogenic  also  for  white  mice,  which  usually 
died  in  six  days  after  being  inoculated  subcutaneously. 

116.   BACILLUS  COPROGENES  FCETIDUS. 

Synonym. — Darmbacillus  of  Schottelius. 

Obtained  by  Schottelius  (1885)  from  the  intestinal  contents  of  pigs  which 
had  died  of  Schweiiierothlauf  (rouget) . 
35 


49b  PATHOGENIC  AEROBIC  BACILLI 

Morphology.  —Resembles  Bacillus  subtilis,  but  is  shorter,  with  rounded 
ends. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motile  bacillus. 
Forms  spores  in  presence  of  oxygen  in  the  course  of  three  or  four  days  at 
the  room  temperature ;  these  are  oval  in  form«and  are  arranged  in  rows; 
when  they  germinate  this  occurs  in  a  direction  perpendicular  to  their  long 
axis  and  to  that  of  the  filament  in  which  they  developed  ;  as  a  result  of 
this  the  newly  formed  rods  lie  parallel  to  each  other.  In  gelatin  stick  cul- 
tures the  growth  upon  the  surface  consists  of  a  thin,  transparent,  grayish 
layer;  along  the  line  of  puncture  crowded,  pale-yellow  colonies  are  de- 
veloped. The  cultures  give  off  an  intense  putrefactive  odor.  'Upon  potato 
a  dry,  grayish  layer  is  formed,  which  may  be  about  0.5  millimetre  in  thick- 
ness. 

Pathogenesis. — Not  pathogenic  for  mice  or  for  rabbits  when  injected  in 
small  amounts,  but  in  considerable  quantities  causes  fatal  toxasmia  in  rabbits. 

117.    BACILLUS  OXYTOCUS  PERNICIOSUS. 

Obtained  by  Wyssokowitsch  from  milk  which  had  been  standing  for  a 
long  time. 

Morphology. — Short  bacilli  with  rounded  ends,  somewhat  thicker  and 
shorter  than  the  lactic  acid  bacillus. 

Biological  Characters. — An  aerobic,  non-liquefying  bacillus.  In  gela- 
tin plates  the  deep  colonies  are  small,  spherical,  finely  granular,  and  of  a 
yellowish  or  brownish-yellow  color.  The  superficial  colonies  are  hemi- 
spherical masses  of  a  grayish- white  color— by  transmitted  light,  light-brown. 
They  may  have  a  diameter  of  one  and  one-half  millimetres. 

In  gelatin  stick  cultures  the  growth  is  at  first  "nail-like"  ;  later  it  ex- 
tends over  the  entire  surface  of  the  gelatin.  It  causes  coagulation  of  milk, 
with  a  sour  reaction,  within  twenty-four  hours.  The  cultures  are  without 
odor. 

Pathogenesis. — Small  doses  are  not  pathogenic  for  mice  or  for  rabbits,  but 
considerable  quantities  injected  into  the  circulation  of  rabbits  cause  their 
death  in  from  three  to  twenty-two  hours.  Soon  after  the  injection  an  abun- 
dant diarrhoea  is  developed.  At  the  autopsy  a  hsemorrhagic  inflammation 
of  the  intestinal  mucous  membrane  is  the  principal  pathological  appearance 
observed. 

118.   BACILLUS  SAPROGENES  II. 

Obtained  by  Rosenbach  (1884)  from  the  perspiration  of  foul-smelling  feet. 

Morphology.  — Short  bacilli  with  rounded  ends. 

Biological  Characters.  — Aerobic  and  facultative  anaerobic.  Characters 
pf  growth  in  gelatin,  motility,  etc. ,  not  given. 

Streak  cultures  upon  the  surface  of  nutrient  agar,  at  the  end  of  twenty- 
four  hours,  cause  the  entire  surface  to  be  covered  with  minute,  transparent 
colonies,  which  later  become  confluent  and  gradually  somewhat  opaque, 
forming  a  viscid,  whitish  gray  layer.  The  odor  of  cultures  resembles  that  of 
perspiring  feet.  Causes  putrefaction  of  albuminous  substances  in  the  pre- 
sence of  oxygen,  with  evolution  of  stinking  gases.  In  the  absence  of  oxygen 
putrefactive  changes  also  occurred,  but  less  rapidly. 

Pathogenesis. — When  injected  in  considerable  quantity  into  the  knee 
joint  or  into  the  pleural  cavity  of  rabbits,  the  animals  succumb  in  from  three 
to  five  days. 

119.    BACILLUS  OF  AFANASSIEW. 

Obtained  by  Afanassiew  (1887)  from  mucus  and  masses  of  pus  coughed 
up  by  patients  suffering  from  whooping  cough.  Etiological  relation  not 
demonstrated. 

Morphology. — Bacilli  from  0.6  to  2.2  ju  long;  solitary,  in  pairs,  or  in 
short  chains. 


NOT    DESCRIBED   IN   PREVIOUS   SECTIONS.  499 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Forms  spores.  Grows  at  the  room  temperature  in  the  usual  culture  media. 
Upon  gelatin  plates  the  colonies  are  spherical  or  oval  and  of  a  light-brown 
color ;  under  a  low  power  they  are  seen  to  be  finely  granular,  and  later  have 
a  dark-brown  color.  Upon  the  surface  of  gelatin  stick  cultures  a  grayish- 
white  layer  is  formed ;  but  slight  development  occurs  along  the  line  of  punc- 
ture. Upon  the  surface  of  agar  a  thick,  gray  layer  forms  along  the  line  of 
inoculation.  Upon  potato  yellowish,  glistening,  dew-like  drops  are  first 
formed  along  the  line  of  inoculation,  and  later  a  rather  thick,  brownish 
layer  is  formed  which  extends  rapidly  over  the  surface.  Development  is 
most  rapid  in  the  incubating  oven. 

Pathogenesis. — According  to  Afanassiew,  pure  cultures  injected  into  the 
air  passages  or  pulmonary  parenchyma,  in  young  dogs  or  in  rabbits,  produce 
bronchial  catarrh,  broncho-pneumonia,  and  attacks  of  spasmodic  coughing 
resembling  those  of  whooping  cough.  Death  sometimes  occurs.  At  the 
autopsy  the  bacillus  is  found  in  great  numbers  in  the  bronchial  and  nasal 
mucus. 

120.    PNEUMOBACILLUS  LIQUEFACIENS  BOVIS. 

Obtained  by  Arloing  from  the  lung  of  an  ox  which  succumbed  to 
infectious  pleuro-pneumonia. 

Morphology- — Slender,  short  bacilli,  which  rather  resemble  mi- 
crococci  when  cultivated  in  gelatin. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic, 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed ;  is 
killed  by  exposure  for  fifteen  to  twenty  minutes  to  a  temperature  of 
55°  C.  Grows  in  the  usual  culture  media  at  the  room  temperature 
— better  at  35°  C.  Forms  white  colonies  in  gelatin  plates,  and 
causes  rapid  liquefaction  of  the  gelatin.  Upon  potato  grows  very 
rapidly  as  a  white  layer,  which  later  has  a  brownish  color. 

Pathogenesis. — From  one-half  to  one  cubic  centimetre  of  a  pure 
culture  injected  beneath  the  skin  of  an  ox,  where  the  connective  tis- 
sue is  loose,  causes  the  development  of  an  acute  abscess  the  size  of  a 
man's  hand ;  after  extending  for  two  or  three  days  this  gradually 
becomes  smaller  and  recovery  occurs.  When  larger  quantities  are 
injected  a  fatal  termination  may  result.  Guinea-pigs  and  rabbits 
are  less  susceptible,  and  dogs  are  said  to  be  immune. 

The  researches  of  Arloing  seeem  to  have  established  the  etiologi- 
cal  relation  of  this  bacillus  to  infectious  pleuro-pneumonia  of  cattle. 
Arloing  has  shown  (1894)  that  inoculations  in  cattle  of  pure  cultures 
of  the  bacillus  are  followed  by  immunity  quite  as  pronounced  as  that 
resulting  from  inoculations  with  serum  from  the  lungs  of  diseased 
animals — method  of  Willems ;  also  that  infected  animals  are  more 
sensible  to  the  action  of  the  toxic  substances  in  filtered  cultures  than 
healthy  animals — corresponding  with  results  obtained  by  use  of  tu- 
berculin and  mallem. 

Arloing  has  obtained  two  varieties  of  his  bacillus  from  the  lungs 
of  cattle,  one  an  attenuated  variety  which  does  not  liquefy  gelatin. 


500  PATHOGENIC  AEROBIC   BACILLI 

He  has  also  found  that  the  liquefying  variety  when  cultivated  in 
bouillon  through  a  series  of  generations  loses  its  liquefying  power  to 
a  considerable  extent.  In  the  liquefying  cultures  the  bacilli  are  often 
elongated  and  articulated ;  in  the  non-liquefying  the  short,  thick  forms, 
with  rounded  ends,  are  most  numerous.  When  injected  in  equal 
quantity  (two  cubic  centimetres)  under  the  skin  of  an  ox  the  results 
are  similar  but  differ  in  degree — the  liquefying  bacillus  producing  a 
more  extensive  local  lesion.  Injected  into  the  lung  the  results  are 
similar :  the  liquefying  bacillus  causes  pneumonic  nodules  as  large 
as  an  apple  and  an  extensive  pleurisy  with  thick  fibrinous  exudation 
infiltrated  with  a  yellow  serum ;  the  non-liquefying  bacillus  causes 
the  development  of  nodules  the  size  of  an  almond  or  of  a  walnut, 
with  a  limited,  but  characteristic,  pleuritic  inflammation. 

Robcis  (1894),  after  discussing  the  results  of  inoculations  made  in 
the  Department  of  the  Seine  with  pulmonary  serum,  arrives  at  the 
conclusion  that  Arloing's  method  of  protective  inoculations  with  cul- 
tures of  the  Pneumobacillus  liquefaciens  bovis  gives  better  results 
than  the  legal  method  with  serum  from  an  infected  animal. 

Arloing  prepares  from  the  cultures  of  his  bacillus  a  "lymph," 
corresponding  with  tuberculin  and  mallein,  which  he  calls  "  pneumo- 
bacilline."  The  toxic  action  of  this  lymph  corresponds  with  that  of 
cultures  sterilized  by  heat.  The  experiments  of  Guinard  and  Artaud 
(1895)  show  that  the  toxic  products  of  the  bacillus  of  Arloing  are 
extremely  active,  and  that  in  dogs  the  injection  of  twenty  to  fifty 
cubic  centimetres  of  a  sterilized  (by  heat)  culture  gives  rise  almost 
immediately  to  torpor  and  sometimes  to  vomiting  and  defecation; 
after  several  hours  vomiting  an$  bloody  diarrhoea  occur,  the  animal 
becomes  more  and  more  feeble,  and  finally,  if  the  dose  has  been  suffi- 
cient, is  completely  paralyzed  and  dies. 

121.    BACILLUS  PSEUDOTUBERCULOSIS. 

Obtained  by  Pfeiffer  (1889)  from  the  organs  of  a  horse  suspected  of  hav- 
ing glanders  and  killed. 

Morphology. — Rather  thick  bacilli  with  round  ends ;  vary  considerably 
in  length — usually  three  to  five  times  as  long  as  broad. 

Stains  with  fuchsin  and  Lotfler's  solution  of  methylene  blue ;  does  not 
stain  by  Gram's  method. 

Biological  Characters.— An  aerobic,  non-liquefying,  non-motile  bacil- 
lus. Spore  formation  not  observed.  Grows  in  the  usual  culture  media  at 
the  room  temperature.  Upon  gelatin  plates,  at  the  end  of  twenty-four 
hours,  the  superficial  colonies  are  small,  yellowish-brown  plates,  which  in- 
crease rapidly  in  diameter;  under  a  low  power  a  central  papilla  is  observed, 
around  which  the  colony  extends  as  a  pale-yellow,  peculiarly  marbled,  crys- 
talline disc  ;  the  deep  colonies  are  at  first  transparent,  sharply  denned  spheres ; 
on  the  third  day,  under  a  low  power,  they  are  seen  to  have  a  dark,  finely 
granular  central  portion  surrounded  by  a  transparent  zone;  when  not 
crowded  upon  the  plate  they  may  appear  as  yellowish-brown,  finely  granu- 
lar, pear-shaped  or  lemon-shaped  colonies.  In  gelatin  stick  cultures  growth 
occurs  along  the  line  of  puncture  in  the  form  of  grayish-white,  spherical 


NOT    DESCRIBED    IN    PREVIOUS    SECTIONS.  501 

colonies,  more  or  less  crowded  above,  and  often  isolated  below,  where  by 
transmitted  light  they  are  seen  to  have  a  brownish  color  ;  upon  the  surface 
a  grayish-white,  concentric  layer  is  formed  about  the  point  of  inoculation  in 
the  course  of  five  or  six  days,  which  later  forms  a  disc  with  thickened  mar- 
gins. Upon  the  surface  of  agar  the  growth  along  the  line  of  inoculation  is- 
abundant  and  viscid.  Does  not  grow  well  upon  potato.  Upon  blood  serum 
forms  transparent,  drop-like  colonies  which  have  an  opalescent  appearance. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  hares,  white  mice, 
and  house  mice.  Death  occurs  in  from  six  to  twenty  days.  At  the  autopsy 
the  lymphatic  glands  are  found  to  be  enlarged  and  to  have  undergone  case- 
ation  ;  the  liver  and  spleen  are  enlarged,  the  lungs  cedematous  and  occasion- 
ally contain  tuberculous-looking  nodules.  An  abscess  forms  at  the  point  of 
inoculation.  Bacilli  are  found  in  the  blood,  the  lymphatic  glands,  and  the 
various  organs. 

122.  BACILLUS   GINGIV^E   PYOGENES. 

Synonym. — Bacterium  gingivae  ^yogenes  (Miller). 

Obtained  by  Miller  from  an  alveolar  abscess  and  from  deposit  around  the 
teeth  "  in  a  filthy  mouth." 

Morphology. — Short  and  thick  bacilli  with  rounded  ends,  one  to  four 
times  as  long  as  broad ;  occur  singly  or  in  pairs. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing  bacillus.  Grows  rapidly  in  the  usual  culture  media.  Upon  gelatin 
plates  it  forms  spherical  colonies  at  the  end  of  twenty-four  hours,  which 
have  a  yellowish  color  and  well-defined  margin ;  at  the  end  of  forty-eight 
hours  liquefaction  has  progressed  so  far  that  the  colonies  have  become  con- 
fluent. In  gelatin  stick  cultures  liquefaction  occurs  rapidly  in  the  form  of  a 
funnel,  at  the  bottom  of  which  a  white  deposit  is  formed.  Upon  the  surface 
of  agar  a  thick,  moist  growth  occurs  along  the  line  of  inoculation,  which 
under  the  microscope  has  a  slightly  greenish-yellow  tint  and  a  fibrillated 
structure. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  and  for  white  mice, 
when  injected  into  the  cavity  of  the  abdomen  in  comparatively  small 
amounts  (0.25  cubic  centimetre).  At  the  autopsy  peritonitis,  sometimes 
purulent,  is  observed.  Death  occurs  in  from  ten  to  twenty-four  hours.  The 
bacilli  are  found  in  the  blood  in  small  numbers.  Subcutaneous  injections  in 
the  animals  mentioned  produce  a  local  abscess  only. 

123.  BACILLUS   DENTALIS   VIRIDANS. 

Found  by  Miller  in  the  superficial  layers  of  carious  dentine. 

Morphology. — Slightly  curved  bacilli  with  pointed  ends;  solitary  or  in 
pairs. 

Biological  CJiaracters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  bacillus.  Spore  formation  not  observed.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  In  gelatin  plates  the  colonies  are 
spherical,  and  under  a  low  power  are  colorless  or  have  a  slightly  yellow  tint; 
when  not  crowded  they  may  present  two  or  three  concentric  rings.  In  gela- 
tin stick  cultures  growth  occurs  both  upon  the  surface  and  along  the  line  of 
puncture.  Gelatin  cultures  acquire  an  opalescent-green  color.  Upon  the 
surface  of  agar  a  thin  growth  with  irregular  margins  occurs  along  the  impf- 
strich ;  this  is  bluish  by  transmitted  light  and  greenish-gray  by  reflected  light 
— colorless  under  the  microscope. 

Pathogenesis. — Injections  into  the  cavity  of  the  abdomen  of  white  mice 
or  of  guinea-pigs  usually  cause  fatal  peritonitis  in  from  one  to  six  days ;  the 
bacilli  are  only  found  in  the  blood  in  small  numbers,  by  the  culture  method. 
Subcutaneous  injections  in  the  animals  mentioned  produce  severe  local  in- 
flammation arid  suppuration. 

124.    BACILLUS   PULP.E   PYOGENES. 
Obtained  by  Miller  from  gangrenous  tooth  pulp. 


502  PATHOGENIC   AEROBIC   BACILLI 

Morphology. — Slightly  curved  bacilli  with  pointed  ends;  solitary  or  in 
pairs,  or  in  chains  of  four  to  eight  elements. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  liquefy- 
ing bacillus.  Spore  formation  not  observed.  Grows  in  the  usual  culture 
media  at  the  room  temperature.  In  gelatin  plates  large,  spherical,  opaque, 
yellowish-brown  colonies  are  formed.  In  gelatin  stick  cultures  liquefaction 
occurs  in  the  upper  part  of  the  tube  and  gradually  extends  downward,  the 
liquefied  gelatin  being  separated  from  the  non-liquefied  by  a  horizontal 
plane. 

Pathogenesis. — Small  quantities  of  a  pure  culture  injected  into  the  abdo- 
minal cavity  of  white  mice  proved  fatal  to  these  animals  in  from  eighteen  to 
thirty  hours. 

125.    BACILLUS   SEPTICUS  KERATOMALACI^. 

Obtained  by  Babes  (1889)  from  the  broken-down  corneal  tissues  and  from 
the  various  organs  of  a  child  which  died  of  septicaemia  following  keratoma- 
lacia. 

Stains  with  the  usual  aniline  colors ;  deeply  colored  granules  may  often 
be  seen  at  the  extremities  of  the  rods,  or  in  the  middle,  in  preparations 
stained  with  Lofner's  solution. 

Morphology. — Short,  thick  bacilli,  thinning  out  at  the  ends;  often  united 
in  pairs ;  may  be  surrounded  by  a  capsule. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying  bacillus.  Spore  formation  not  observed.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  Upon  gelatin  plates  forms  white, 
slightly  elevated,  flat  colonies  with  finely  dentate  margins.  In  gelatin  stick 
cultures  the  growth  is  abundant  both  on  the  surface  and  aiong  the  line  of 
puncture ;  gas  bubbles  are  formed  in  the  gelatin.  Upon  the  surface  of  agar 
the  growth  along  the  line  of  inoculation  is  leaf-like,  finely  dentate,  some- 
what opalescent,  and  the  culture  has  a^slightly  ammoniacal  odor.  Upon 
blood  serum  a  semi-transparent,  glistening  film  is  formed,  which  has  dentate 
margins. 

Pathogenesis. — Pathogenic  for  rabbits  and  mice,  less  so  for  birds;  not 
pathogenic  for  guinea-pigs.  The  animals  die  in  from  three  to  seven  days. 
Inoculated  into  the  cornea  it  causes  a  purulent  keratitis. 

126.    BACILLUS   SEPTICUS  ACUMINATUS. 

Obtained  by  Babes  (1889)  from  the  blood,  the  umbilical  stump,  and  the 
various  organs  of  a  child  which  died  five  days  after  birth,  apparently  from 
septic  infection. 

Morphology.  — Bacilli  with  lancet-shaped  ends,  somewhat  resembling  the 
bacillus  of  mouse  septicaemia,  but  thicker.  Often  shows  unstained  places  in 
the  middle  of  the  rods  in  stained  preparations. 

Stains  readily  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic  bacillus;  does  not  grow  in  gelatin  at 
the  room  temperature.  Spore  formation  not  observed.  Grows  upon  blood 
serum  and  upon  nutrient  agar  at  37°  C.,  in  form  of  small,  flat,  circular, 
transparent,  shining  colonies,  which  become  confluent  and  later  form  a  yel- 
lowish layer.  Blood  serum  is  the  most  favorable  medium. 

Pathogenesis. — Pathogenic  for  rabbits  and  guinea-pigs,  not  for  mice. 
The  animals  die  in  from  two  to  six  days,  and  the  bacilli  are  found  in  their 
blood  u ml  in  the  various  organs. 

127.    BACILLUS   SEPTICUS  ULCERIS   GAXGR^ENOSI. 

Obtained  by  Babes  (1889)  from  the  blood  and  various  organs  of  a  boy  \vli<> 
died  from  septicaemia  following  gangrene  of  the  skin,  etc. 

Morphology. — Bacilli  with  round  ends,  oval  or  rod-shaped,  about  0.5  to 
0.6  u  thick. 


I  NOT   DESCRIBED   IN  PREVIOUS   SECTIONS.  503 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacillus.  Does 
not  form  spores.  Grows  in  the  usual  culture  media  at  the  room  temperature. 
In  gelatin  stick  cultures  a  sac-formed  liquefaction  occurs  and  a  yellow  de- 
posit is  seen  at  the  bottom  of  the  liquefied  gelatin ;  gas  bubbles  are  given  off 
from  the  culture.  Upon  the  surface  of  agar  development  occurs  along  the 
line  of  inoculation  in  the  form  of  flat,  grayish-yellow,  transparent,  varnish- 
like  plaques.  Upon  potato,  after  several  days,  a  brownish,  shining,  moist, 
transparent  film  is  formed.  Upon  the  surface  of  blood  serum  smooth, 
yellowish,  transparent  colonies  are  formed,  under  which  the  blood  serum  is 
softened,  allowing  these  to  sink  below  the  surface. 

Pathogenesis. — Pathogenic  for  mice  and  for  guinea-pigs,  which  die  in 
from  one  to  two  days.  An  abscess  forms  at  the  point  of  inoculation,  which 
is  covered  with  a  dry,  retracted  crust. 

128.    BACILLUS   OF  TRICOMI. 

Obtained  by  Tricomi  (1886)  from  a  case  of  senile  gangrene. 

Morphology. — Bacilli  with  round  ends,  about  three  /*  long  and  one  u 
thick,  solitary  o-  in  pairs ;  sometimes  one  end  of  a  rod  shows  a  club-shaped 
thickening. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 
*  Biological  Characters.  -An    aerobic,    liquefying,  non-motile  bacillus. 
Forms  spores.     Grows  in  the  usual  culture  media  at  the  room  temperature — 
better  at  37°  C.  f 

Upon  gelatin  plates,  at  the  end  of  twenty-four  hours,  the  colonies  are 
spherical,  finely  granular,  and  of  a  dirty-yellow  color  ;  after  from  thirty.-six 
to  forty-eight  hours  liquefaction  of  the  surrounding  gelatin  occurs.  In  gela- 
tin stick  cultures  closely  crowded,  small,  white  colonies  are  formed  along 
the  line  of  puncture  ;  at  the  end  of  forty-eight  hours  liquefaction  com- 
mences in  funnel  form,  with  formation  of  an  air  bubble  above — like  the 
cholera  spirillum;  later  the  entire  gelatin  is  liquefied  and  becomes  trans- 
parent, while  a  dirty-white  collection  of  bacilli  is  seen  at  the  bottom  of  the 
tube.  Upon  the  surface  of  agar  a  white  layer  with  irregular  margins  is 
formed,  which  later  extends  over  the  entire  surface  as  a  homogeneous,  rather 
thin  membranous  film.  \Jyon potato,  at  37°  C.,  dirty- white,  milky  colonies 
are  formed,  which  later  become  confluent.  Upon  blood  serum  the  growth  is 
similar  to  that  upon  agar. 

Pathogenesis. — The  subcutaneous  injection  of  one-half  to  one  cubic  centi- 
metre of  a  gelatin  culture  is  said  by  Tricomi  to  produce  in  rabbits  and  in 
guinea-pigs  a  gangrenous  process  resembling  senile  gangrene  in  man.  The 
subcutaneous  connective  tissue  is  infiltrated  with  a  foul-smelling  serum,  the 
muscles  are  soft  and  gray,  and  a  portion  of  the  skin  has  a  mummified  ap- 
pearance. The  gangrene  extends  over  the  abdomen,  and  death  occurs  in 
guinea-pigs  in  two  to  three  days,  in  rabbits  after  four  days,  in  house  mice 
at  the  end  of  twenty-four  hours ;  white  mice  are  said  to  be  immune. 

129.    BACILLUS   ALBUS   CADAVERIS. 

Obtained  by  Strassmann  and  Strieker  (1888)  from  the  blood  of  two  cada- 
vers four  days  after  death. 

Morphology.— Bacilli  about  two  and  one-half  /*  long  and  0.75  fi  broad; 
also  grow  out  into  filaments  of  six  fit  or  longer. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters.— Anaerobic,  liquefying,  motile  bacillus.  Spore 
formation  not  observed.  Grows  in  the  usual  culture  media  at  the  room  tem- 
perature. In  gelatin  plates  small,  spherical,  yellowish  colonies  are  formed 
during  the  first  twenty-four  hours ;  later  a  radiating  outgrowth  occurs  from 
the  periphery,  and  liquefaction  of  the  gelatin  takes  place.  In  gelatin  stick 
cultures  liquefaction  begins  within  forty-eight  hours,  and  forms  a  long  fun- 
nel, at  the  opening  of  which  is  a  cavity  containing  air  ;  the  liquefied  gela- 


504  PATHOGENIC   AEROBIC   BACILLI 

tin  is  transparent,  and  a  deposit  of  thick,  granular  masses  accumulates  at  the 
bottom  of  the  tube.  Upon  the  surface  of  agar  a  thick,  white  layer  is  formed, 
which  later  is  wrinkled  and  after  a  time  gives  off  a  putrefactive  odor.  Gela- 
tin cultures  give  off  an  odor  of  sulphuretted  hydrogen.  Upon  potato  a  soft, 
white  or  pale-yellow  layer  is  formed,  which  in  places  is  made  up  of  small 
granules.  The  potato  around  the  growth  has  a  bluish-brown  color. 

Pathogenesis. — Subcutaneous  injection  of  a  small  quantity  (0.1  cubic 
centimetre)  of  a  liquefied  gelatin  culture  is  fatal  to  mice  in  about  six  hours ; 
the  animals  become  comatose  before  death,  and  at  the  autopsy  putrefactive 
changes  are  already  observed ;  the  bacillus  can  be  recovered  from  the  blood 
in  cultures.  Sterilized  cultures  also  prove  fatal  to  mice.  Pathogenic  also 
for  guinea-pigs,  which  die  in,  about  twenty  hours  after  receiving  a  subcuta- 
neous inoculation. 

130.    BACILLUS  VARICOSUS   CONJUNCTIVE. 

Obtained  by  Gombert  (1889)  from  the  healthy  conjunctival  sac  of  man. 

Morphology. — Large  bacilli  with  round  ends,  from  two  to  eight  n  long 
and  about  one  JJL  broad;  the  shorter  bacilli  are  often  constricted  in  the 
middle. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, non-motile  bacillus.  Grows  very  slowly  in  nutrient  gelatin  at  22°  C. ; 
rapidly  in  agar  and  upon  potato  at  37°  C.  In  gelatin  stick  cultures,  at  the 
end  of  twenty-four  hours,  a  circular  layer  haying  a  grayish- white  centre  is 
developed  upon  the  surface,  and  a  scarcely  visible  grayish- white  thread  along 
the  line  of  puncture.  Liquefaction  extends  gradually  from  the  surface 
without  clouding  or  changing  the  gelatin,  so  that  at  the  end  of  two  weeks 
the  gelatin  is  entirely  liquefied  without  giving  any  other  evidence  of  the  pre- 
sence of  the  microorganism.  Upon  agar  plates,  at  37°  C.,  the  deep  colonies 
have  a  diameter  of  about  four  millimetres  by  the  end  of  the  fourth  day; 
under  a  low  power  they  are  seen  to  be  covered  with  minute,  irregular,  thorn- 
like  projections,  which  subsequently  increase  in  size ;  the  centre  of  the  colony 
is  granular  and  opaque.  The  superficial  colonies,  under  a  low  power,  are  seen 
to  have  an  opaque  central  nucleus  surrounded  by  a  yellowish,  finely  granu- 
lar, transparent  peripheral  zone;  later  the  central  portion  is  irregular  and 
semi-opaque,  surrounded  by  a  broad  marginal  zone  which  consists  of  twisted 
and  bent  tapering  offshoots  having  a  dark  contour.  Upon  the  surface  of 
agar  a  thin,  white,  dry,  very  adherent  film  is  formed ;  a  thick,  white  film 
forms  upon  the  surface  of  the  condensation  water.  Upon  potato  develop- 
ment is  rapid  at  37°  C.,  forming  at  first  a  dry,  white  layer,  which  at  the  end 
of  ten  days  covers  the  entire  surface ;  it  then  has  an  irregular  surface  and 
fringed  margins,  is  smooth,  dry,  and  after  a  time  has  a  reddish-brown  color. 

Pathogenesis. — When  inoculated  into  the  cornea  of  rabbits  a  grayish- 
white  cloudiness  is  developed  in  twenty-four  hours,  around  which  the  cornea 
is  highlv  vascular ;  the  animal  recovers  without  the  formation  of  an  abscess. 
Injected  into  the  conjunctiva  it  causes  an  intense  hyperaemia. 

131.    BACILLUS   MENINGITIDIS   PURULENTJE. 

Obtained  by  Neumann  and  Schaffer  (1887)  from  pus  from  beneath  the  pia 
mater  in  an  individual  who  died  of  purulent  meningitis. 

Morphology. — Bacilli  about  two  u  long  and  0.6  to  0.7/*  broad;  often 
grow  out  into  long  filaments,  especially  in  gelatin  cultures. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Does  not  form  spores.  Grows  in  the  usual 
culture  media  at  the  room  temperature — better  in  the  incubating  oven.  Upon 
gelatin  plates  the  deep  colonies,  under  a  low  power,  are  homogeneous,  round 
or  oval,  pale  brown,  and  with  a  smooth  contour;  the  superficial  colonies  are 


NOT   DESCRIBED   IX   PREVIOUS   SECTIONS.  505 

thin,  moist,  and  transparent  in  appearance;  later  they  have  a  grayish  color, 
a  coarsely  granular  surface,  and  are  made  up  of  flap-like  layers.  In  gelatin 
stick  cultures  the  superficial  growth  consists  of  broad,  grayish  layers,  and  a 
grayish-yellow  growth  is  seen  along  the  line  of  puncture,  made  up  of  crowded 
colonies.  Upon  agar  plates,  at  the  end  of  twenty-four  hours  at  37°  C., 
thin  colonies  are  developed,  which  have  a  granular  surface,  a  smooth,  more 
or  less  irregular  outline,  and  a  pale-brown  color  in  the  centre.  Upon  potato 
a  scanty,  moist,  white  layer  develops  along  the  line  of  inoculation.  Upon 
blood  serum,  at  37°  C. ,  at  the  end  of  twenty-four  hours  a  moist,  shining  layer 
about  four  millimetres  broad  is  developed  along  the  impf  strich ;  this  is  gra- 
nular at  the  margins,  and  later  more  or  less  fissured. 

Pathogenesis.  —Subcutaneous  injection  produces  in  dogs,  rabbits,  guinea- 
pigs,  and  white  mice  a  purulent  inflammation  in  the  vicinity  of  the  point  of 
inoculation. 

132.    BACILLUS   SEPTICUS  VESIOE. 

Obtained  by  Clado  (1887)  from  the  urine  of  a  person  suffering  from  cys- 
titis. 

Morphology. — Bacilli  with  round  ends,  1  6  to  2  ji  long  and  0.5  jn  thick; 
never  united  in  pairs  or  chains. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Forms  spores.  Grows  in  the  usual  culture 
media  at  the  room  temperature.  Upon  gelatin  plates  small,  spherical  or 
oval  colonies  are  developed  throughout  the  gelatin,  which  rarely  exceed  the 
size  of  a  pin's  head  ;  these  are  transparent,  and  yellowish-white  in  color  ; 
under  a  low  power  the  centre  is  seen  to  be  dark  gray  and  is  surrounded  by  a 
well-defined  marginal  zone  of  a  pale-yellow  color.  In  gelatin  stick  cultures 
the  growth  along  the  line  of  puncture  is  first  seen  as  a  delicate,  whitish 
thread  ;  at  the  end  of  six  or  seven  days  it  is  made  up  of  lenticular  colonies, 
one-third  as  large  as  a  pin's  head,  arranged  in  two  lines  like  piles  of  coin. 
Upon  the  surface  the  growth  is  scanty  and  consists  of  a  thin  layer  around  the 
point  of  inoculation,  which  has  a  jagged  contour.  Upon  the  surface  of  agar 
development  is  slow  and  forms  a  grayish- white  stripe  along  the  impf  strich. 
Upon  potato  a  flat,  dry,  chestnut-brown  layer  is  formed. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  and  mice.  Death 
appears  to  result  from  the  toxic  products  formed,  as  well  as  from  the  multi- 
plication of  the  bacilli  in  the  inoculated  animals. 

133.    BACILLUS   OF   GESSNER.' 

Synonym. — Bacterium  tholoideum  (Gessner). 

Obtained  by  Gessner  from  the  contents  of  the  intestine  of  healthy  persons. 
Resembles  in  its  morphology  and  in  its  growth  in  culture  media  Bacillus 
lactis  aerogenes  of  Escherich. 

Pathogenic  for  mice  and  for  guinea-pigs. 

134.    BACILLUS   CHROMO-AROMATICUS. 

Obtained  by  Galtier  (1888)  from  a  pig  which  died  from  a  general  infec- 
tious malady  characterized  by  broncho-pneumonia,  pleuritis,  enteritis,  and 
swelling  of  the  lymphatic  glands. 

Morphology. — -Bacilli  of  medium  size  with  rounded  ends. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Not  observed  to  form  spores.  Grows  in  the  usual  cul- 
ture media  at  the  room  temperature — better  in  the  incubating  oven.  The 
cultures  all  produce  a  green  or  brown  pigment  and  have  an  aromatic  odor. 
In  gelatin  stick  cultures  a  yellowish-white  layer  is  formed  upon  the  surface 
of  the  liquefied  gelatin,  which  has  a  bright-green  color  ;  a  yellowish-white 


506  PATHOGENIC   AEROBIC   BACILLI 

deposit  accumulates  at  the  bottom  of  the  tube.  Upon  the  surface  of  agar 
whitish  colonies  are  formed,  which  coalesce  to  form  a  thin  layer.  Upon 
potato  a  tolerably  thick,  somewhat  iridescent,  brown  layer  is  formed,  which 
extends  over  the  entire  surface.  In  bouillon,  at  the  end  of  twenty-four  to 
forty-eight  hours  at  37°  C.,  a  greenish-yellow  color  is  developed,  first  near 
the  surface  and  later  extending  throughout  the  fluid,  which  acquires  the  color 
of  a  dilute  solution  of  sulphate  of  copper  ;  a  whitish  film  forms  upon  the 
surface.  In  anaerobic  cultures  the  color  is  a  pale  brown  instead  of  green. 

Pathogenesis.  — Rabbits  die  at  the  end  of  two  to  three  weeks  after  receiv- 
ing an  intravenous  injection.  At  the  autopsy  they  are  found  to  have  pneu- 
monia with  pleuritis  and  pericarditis. 

135.    BACILLUS  CANALIS  CAPSULATUS. 

Obtained  by  Mori  (1888)  from  sewer  water. 

?  Morphology. — Bacilli  with  round  ends,  elliptical  or  rod-shape  in  form, 
and  from  0.9  to  1.6  jn  thick  ;  often  surrounded  with  a  broad  capsule,  which 
is  always  seen  in  preparations  from  the  blood  or  tissues  of  an  infected  ani- 
mal ;  sometimes  in  pairs  with  the  acute  ends  of  the  rods  in  apposition,  and 
surrounded  by  a  single  capsule. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters.—  An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates 
hemispherical,  porcelain- white,  sharply  defined  colonies,  resembling  those  of 
Friedlander's  bacillus,  are  developed  at  the  end  of  twenty -four  hours.  In 
gelatin  stick  cultures  development  occurs  along  the  line  of  puncture  and 
upon  the  surface,  forming  a  u  nail-shaped  "  growth  similar  to  that  of  Fried- 
lander's  bacillus  (Bacillus  pneumonise)  in  the  same  medium.  Upon  agar 
a  viscid  and  abundant  growth  is  formed  in  the  incubating  oven  at  37°  C. 
"Upon  potato  an  abundant  development  in  the  form  of  a  yellowish,  moist,  vis- 
cid layer,  with  irregular  outlines.  In  bouillon,  at  the  end  of  three  or  four 
days,  a  white  film  forms  on  the  surface,  especially  in  contact  with  the  test 
tube. 

Pathogenesis. — Mice  die  in  two  to  three  days  after  receiving  a  subcutane- 
ous injection.  Guinea-pigs  and  rabbits  are  immune. 

136.  BACILLUS  CANALIS  PARVUS. 

Obtained  by  Mori  (1888)  from  sewer  water. 

Morphology. — Bacilli  with  round  ends,  from  2  to  5  n  long  and  0.8  to  1  ft 
broad. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method  ;  the 
ends  of  the  rods  are  more  deeply  stained  than  the  central  portion. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motile  ba<-il 
lus.  Not  observed  to  form  spores.  Grows  very  slowly  at  the  room  tempera- 
ture— more  rapidly  at  37°  C.  Upon  gelatin  plates,  at  the  end  of  two  to  three 
weeks,  extremely  minute,  homogeneous,  pale-yellow  colonies  are  developed. 
In  gelatin  stick  cultures  a  thin,  yellowish  layer  forms  upon  the  surface  at 
the  end  of  three  weeks.  Upon  the  surface  of  agar,  at  37°  C. ,  a  dry,  yellow- 
ish layer  with  jagged  outlines  is  developed  in  two  or  three  days.  No  growth 
occurs  upon  potato.  Upon  blood  serum  a  thin,  pale-green,  dry  layer  is 
formed. 

Pathogenesis.—  Mice  die  in  from  sixteen  to  thirty  hours  after  receiving  a 
subcutaneous  inoculation,  guinea-pigs  in  about  two  days. 

137.    BACILLUS  INDIGOGENUS. 

Obtained  by  Alvarez  (1887)  from  an  infusion  of  the  leaves  of  the  indigo 
plant. 

Morphology. — Bacilli  with  round  ends,  about  3  //  long  and  1.5  /*  thick, 


NOT  DESCRIBED   IN  PREVIOUS   SECTIONS.  507 

often  united  in  chains  of  six  to  eight  elements.  The  cells  are  surrounded  by 
a  transparent  capsule  resembling  that  of  Friedlander's  bacillus. 

Biological  Characters. — An  aerobic,  motile  bacillus.  Upon  agar,  at 
37°  C. ,  a  yellowish- white  layer  is  quickly  developed  and  there  is  production 
of  gas.  According  to  Alvarez,  this  bacillus  develops  an  indigo-blue  color  in 
a  sterilized  infusion  of  the  leaves  of  the  indigo  plant. 

Pathogenesis. — Guinea  pigs  die  in  from  three  to  twelve  hours  from  the 
intravenous  injection  of  a  pure  culture. 

138.   BACILLUS  OF  KARTULIS. 

Obtained  by  Koch  (1883)  and  by  Kartulis  from  the  conjunctiyal  secre- 
tions of  persons  suffering  from  a  form  of  infectious  catarrhal  conjunctivitis 
which  prevails  in  Egypt. 

Morphology. — Resembles  the  bacillus  of  mouse  septicaemia  (Bacillus  mu- 
risepticus)  in  its  form  and  dimensions. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic  bacillus.  Does  not  grow  in  nutri- 
ent gelatin  at  the  room  temperature.  Upon  the  surface  of  nutrient  agar,  at 
28°  to  30°  C.,  at  the  end  of  thirty  to  forty  hours  small,  grayish- white  points 
are  developed  along  the  impfstrich  ;  later  these  become  confluent  and  form 
an  elevated,  shining,  dark-colored  layer  with  irregular  and  often  jagged 
margins. 

Pathogenesis. — Out  of  six  experimental  inoculations,  with  pure  cultures, 
made  by  Kartulis  in  the  eyes  of  healthy  individuals,  four  gave  a  negative 
result,  one  produced  a  catarrhal  inflammation  lasting  for  a  week,  in  an  e^e 
which  was  blind  from  a  previous  attack  of  sclerochoroiditis,  and  one  a  con- 
junctivitis lasting  for  ten  days  in  a  perfectly  healthy  eye. 

139.   BACILLUS  OF  UTPADEL. 

Obtained  by  Utpadel  (1887)  from  the  wards  of  a  military  hospital  at  Augs- 
burg— in  the  ' '  Zwischendeckenf iillung  " ;  also  by  Gessner  from  the  contents 
of  the  small  intestine  in  man. 

Morphology. — Bacilli  with  round  ends,  1.25  to  1.5  n  long  and  0.75  to  1  /* 
thick ;  often  united  in  pairs  or  in  chains  of  three  elements. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Grows  in  the  usual  culture  media  at  the  room  temperature.  Spore  forma- 
tion not  observed.  Upon  gelatin  plates  the  superficial  colonies  are  elevated 
and  sometimes  conical,  and  of  a  milk-white  color.  The  deep  colonies  are 
round  or  oval ;  the  centre  is  dark  green  and  is  surrounded  by  a  brownish- 
green  peripheral  zone.  Upon  the  surface  of  agar  a  yellowish-white  layer 
is  developed  very  slowly.  The  growth  upon  gelatin  is  rapid. 

Pathogenesis. — When  injected  subcutaneously  into  cats,  guinea-pigs,  or 
mice  it  produces  an  extensive  inflammatpry  oedema,  resulting  in  the  death 
of  the  animals. 

140.    BACILLUS  ALVEI. 

Synonym. — Bacillus  of  foul  brood  (of  bees). 

Obtained  by  Cheshire  and  Cheyiie  (1885)  from  the  larvae  in  hives  infected 
with  "  foul  brood."  The  larvae  in  the  interior  of  cells  in  the  comb  die  and 
become  almost  fluid  as  a  result  of  parasitic  invasion  by  this  bacillus. 

Morphology. — Bacilli  with  rounded  ends,  from  2.5  to  5  ju  in  length  (aver- 
age about  3.6  ju)  and  0.8  u  in  diameter.  Grow  out  into  filaments  and  form 
large  oval  spores  which  have  a  greater  diameter  than  the  rods  in  which  they 
are  developed — 1.07  n. 

Stains  readily  with  the  aniline  colors  usually  employed,  also  by  Gram's 
method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 


508  PATHOGENIC   AEROBIC   BACILLI 

ing,  motile  bacillus.  Forms  endogenous  spores.  Grows  readily  in  the  usual 
culture  media  at  the  room  temperature. 

In  gelatin  plates  small,  round  or  oval  colonies  are  formed,  which  later 
become  pear-shaped ;  a  branching  outgrowth  occurs  about  the  margins  of  the 
colonies,  and  especially  from  the  small  end  of  the  pear-shaped  mass.  In 
streak  cultures  upon  the  surface  of  gelatin  growth  occurs  first  along  the  impf- 
strich,  and  from  this  an  outgrowth  occurs  consisting  of  bacilli  in  a  single 
row  or  in  several  parallel  rows,  and  forming  irregular  or  circular  figures, 
from  which  other  similar  outgrowths  occur;  the  branching  outgrowths  may 
anastomose.  The  gelatin  is  liquefied  in  the  vicinity  of  these  lines  of  growth, 
forming  a  network  of  channels.  A  similar  growth  is  seen  upon  the  surface 
of  gelatin  stick  cultured,  and  along  the  line  of  puncture  white,  irregular 
masses  are  formed,  from  which  rather  coarse  branches  are  given  off  which 
often  have  a  club-shaped  extremity.  In  older  cultures  the  finer  branches 
disappear,  so  that  the  secondary  centres  of  growth  are  disconnected  from  the 
original  colonies ;  complete  liquefaction  of  the  gelatin  occurs  in  about  two 
weeks;  the  liquefied  gelatin  has  a  yellowish  color  and  peculiar  odor.  Upon 
the  surface  of  nutrient  agar,  at  37°  C. ,  a  white  layer  is  formed.  Upon  potato 
the  development  is  slow  and  results  in  the  formation  of  a  dry,  yellowish 
layer.  In  milk  coagulation  first  occurs,  and  the  coagulum  is  subsequently 
dissolved;  a  slightly  acid  reaction  is  produced.  This  bacillus  grows  best  in 
the  incubating  ovea  at  37°,  and  does  not  develop  at  temperatures  below  16° 
C.  The  spores  require  for  their  destruction  a  temperature  of  100°  C.  main- 
tained for  four  minutes  (determined  by  the  writer,  1887). 

Pathogenesis. — The  introduction  of  pure  cultures  of  this  bacillus  into 
hives  occupied  by  healthy  swarms  causes  them  to  become  infected  with  foul 
brood;  grown  bees  also  become  infected  when  given  food  containing  the  ba- 
cillus (Cheshire) .  Mice  injected  subcutaneously  with  a  considerable  quan- 
tity die  within  twenty-four  hours,  guinea-pigs  in  six  days  (Eisenberg). 
Small  amounts  injected  beneath  the  skin  of  mice  or  rabbits  produce  no  appa- 
rent result. 

141.  BACILLUS  OP  ACNE  CONTAGIOSA  OF  HORSES. 

Obtained  by  Dieckerhoff  and  Grawitz  (1885)  from  pus  and  dried  scales 
from  the  pustules  of  "  acne  conta^iosa  "  of  horses. 

Morphology.— Short  rods,  straight  or  slightly  bent,  0.2  ju  in  diameter. 

Stains  best  with  an  aqueous  solution  of  fuchsin,  and  also  by  Gram's 
method ;  does  not  stain  well  with  Loftier 's  alkaline  solution  of  methvlene 
blue. 

Biological  Characters. — Anaerobic,  non-liquefying  bacillus.  In  gelatin 
stick  cultures  a  very  scanty  growth  occurs  along  the  line  of  puncture ;  upon 
the  surface  a  white  mass  forms  about  the  point  of  puncture.  Upon  blood 
serum  and  nutrient  agar  an  abundant  growth  at  the  end  of  twenty-four 
hours  at  37°  C.,  consisting  of  white  colonies  along  the  impfstrich,  which 
later  have  a  yellowish-gray  color.  The  growth  is  more  abundant  and  rapid 
upon  blood  serum  than  upon  other  media. 

Pathogenesis.  — Pure  cultures  of  the  bacillus  described  are  said  by  Diecker- 
hoff and  Grawitz  to  produce  typical  acne  pustules  when  rubbed  into  the  skin 
of  horses,  calves,  sheep,  and  dogs.  When  rubbed  into  the  intact  skin  of 
guinea-pigs  a  phlegmonous  erysipelatous  inflammation  was  produced,  and 
the  animal  died  at  the  end  of  forty-eight  hours  with  symptoms  of  toxemia. 
Subcutaneous  injections  in  guinea-pigs  caused  toxaemia  and  death  at  the  end 
of  twenty-four  hours.  A.t  the  autopsy  a  haemorrhage  infiltration  of  the  in- 
testinal mucous  membrane  was  observed ;  the  bacilli  were  not  found  in  the 
internal  organs.  In  rabbits  pure  cultures  rubbed  into  the  intact  skin  caused 
a  development  of  pustules  and  a  severe  inflammation  of  the  subcutaneous 
connective  tissue,  t'n  >i 1 1  w  1 1  ich  the  animal  usually  recovered.  Subcutaneous 
injections  in  rabbits  sometimes  caused  a  fatal  toxiemia.  House  mice,  field 
mice,  and  white  mice  were  not  affected  by  the  application  of  cultures,  by 


NOT   DESCRIBED   IN  PREVIOUS   SECTIONS.  509 

rubbing,  to  the  uninjured  skin,  but  succumbed  to  subcutaneous  injections  in 
twenty-four  hours  or  between  the  fifth  and  tenth  days.  Those  which  died 
at  a  late  date  presented  the  pathological  appearances  which  characterize 
pysemia. 

142.   BACILLUS  NO.    I  OF  ROTH. 

Obtained  by  Roth  (1890)  from  old  rags.  Resembles  Bacillus  coli  com- 
munis  and  Brieger's  bacillus  in  its  morphology  and  growth  in  various  culture 
media,  but,  according  to  Roth,  is  distinguished  from  these  bacilli  by  the  fact 
that  colonies  upon  gelatin  plates  are  thicker  and  more  opaque. 

Pathogenesis. — Pathogenic  for  rabbits  and  for  guinea-pigs  when  injected 
into  the  cavity  of  the  abdomen;  death  usually  occurs  within  twenty-four 
hours.  The  spleen  is  greatly  enlarged,  and  the  bacilli  are  found  in  cultures 
from  the  blood  and  various  organs. 

143.    BACILLUS  NO.    II  OF  ROTH. 

Obtained  by  Roth  (1890)  from  old  rags. 

Morphology. — Bacilli  with  round  ends,  0.6  to  1  jo,  broad  and  two  to  four 
times  as  long. 

Stains  with  the  usual  aniline  colors.  When  stained  by  Gram's  method 
it  is  decolorized  by  alcohol. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Grows  in  the  usual  culture  media  at  the 
room  temperature.  Upon  gelatin  plates  colonies  resembling  those  of  the 
<;olon  bacillus  are  developed  at  the  end  of  twenty-four  hours ;  on  the  third 
day  small,  drop-like,  shining,  bluish-white  colonies,  around  the  periphery  of 
which  a  commencing  extension  upon  the  surface  of  the  gelatin  is  seen.  Older 
colonies  are  seldom  more  than  one-half  centimetre  in  diameter,  and  are  some- 
what thicker  than  this ;  they  are  nearly  transparent.  Upon  the  surface  of 
gelatin  stick  cultures  a  rather  moist,  yellowish-white  layer  with  dentate 
margins  is  developed.  Upon  potato  a  colorless  layer  is  developed,  which 
later  has  a  grayish  color. 

Pathogenic  for  rabbits  and  guinea-pigs  when  injected  into  the  abdominal 
cavity. 

144.   BACILLUS  OF  OKADA. 

Obtained  by  Okada  (1891)  from  dust  between  the  boards  of  a  floor. 

Morphology. — Short  rods  with  round  ends,  about  as  long  as  Bacillus 
murisepticus,  but  somewhat  thicker — about  twice  as  long  as  thick ;  solitary 
or  in  pairs;  in  old  cultures  may  grow  out  into  filaments. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  Upon  gelatin  plates,  at  the  end  of 
two  to  three  days,  small,  white,  spherical  colonies  are  developed.  Under 
the  microscope  these  are  seen  to  be  granular,  pale-brown  in  color,  and  with 
slightly  jagged  margins ;  the  superficial  colonies  after  several  days  are  con- 
siderably elevated  above  the  level  of  the  gelatin.  In  gelatin  stick  cultures 
development  occurs  as  a  white  thread  along  the  line  of  puncture,  and  upon 
the  surface  as  a  flat,  milk-white  layer  which  does  not  extend  to  the  walls  of 
the  test  tube.  Upon  agar,  at  37°  C.,  the  growth  is  rapid  and  the  surface  is 
nearly  covered  at  the  end  of  eighteen  hours  with  a  milk-white  layer ;  the  con- 
densation water  is  filled  with  a  viscid  mass  of  bacilli.  Upon  blood  serum 
the  growth  is  shining  and  almost  transparent.  In  bouillon  development  is 
rapid,  clouding  the  fluid  throughout,  and  a  cream-like  layer  forms  upon  the 
surface. 

Pathogenesis. — Rabbits  and  guinea-pigs  die  in  about  twenty  hours  after 
receiving  a  subcutaneous  injection  of  a  half-syringeful  of  a  bouillon  cul- 
ture, or  from  a  small  quantity  (two  ose)  from  a  gelatin  or  agar  culture.  In 


510  PATHOGENIC   AEROBIC   BACILLI 

mice  a  minute  quantity  of  a  pure  culture  invariably  proved  fatal  in  about 
twenty  hours.  Four  hours  after  the  inoculation  an  abundant  secretion  from 
the  lachrymal  glands  occurs,  and  soon  after  the  eyes  become  completely  closed. 
According  to  Okada,  this  bacillus  is  differentiated  from  the  bacillus  of 
Briefer,  and  from  Emmerich's  bacillus  which  it  greatly  resembles,  by  the 
fact  that  it  does  not  grow  upon  potato. 

145.    BACILLUS  OF  PURPURA  H^MORRHAGICA  OF  TIZZONI  AND 

GIOVANNINI. 

Obtained  by  Tizzoni  and  Giovannini  (1889)  from  the  blood  of  two  children 
who  died  of  purpura  haemorrhagica  following  impetigo. 

Morphology.—  Bacilli  with  round  ends,  from  0.75  to  1.3  fit  long  and  0.2 
to  0.4  fj.  broad;  often  seen  in  pairs  or  in  groups  like  streptococci. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  the 
colonies  at  first  resemble  those  of  Streptococcus  pyogenes.  Upon  the  surface 
small,  opaque  points  are  seen  at  the  end  of  forty-eight  hours,  which  at  the 
end  of  four  to  five  days  develop  into  spherical,  yellowish-gray  colonies  with 
irregular  margins,  surrounded  by  a  growth  resembling  tufts  of  curly  hair. 
Upon  agar  the  growth  is  similar,  but  more  rapid  and  of  a  pale  color,  often 
with  a  central  nucleus  surrounded  by  a  net-like  marginal  zone.  Upon 
blood  serum  the  growth  is  similar  to  that  upon  agar.  Upon  potato,  at  37° 
C.,  a  limited  development  occurs  about  the  point  of  inoculation,  which  has 
a  dark-yellow  color.  The  cultures  give  off  a  very  penetrating  odor. 

Pathogenesis. — Pathogenic  for  dogs,  rabbits,  and  guinea-pigs  when  in- 
jected subcutaiieously.  Not  pathogenic  for  white  mice  or  pigeons.  The 
symptoms  resulting  from  a  subcutaneous  injection  are  said  to  be  fever,  al- 
buminuria  and,  in  some  cases,  anuria,  haemorrhagic  spots  upon  the  skin, 
convulsions  :  death  occurs  in  from  one  to  three  days.  At  the  autopsy  there 
are  found  oedema  about  the  point  of  inoculation,  haemorrhages  in  the  skin  and 
muscles,  and  sometimes  in  the  internal  organs  and  in  serous  cavities;  the 
blood  does  not  coagulate.  The  bacilli  are  found  in  the  subcutaneous  con- 
nective tissue,  but  not  in  the  blood  or  in  the  various  organs.  Sections  show 
coagulation  necrosis  of  the  liver  cells  and  of  the  renal  epithelium. 

146.    BACILLUS  OF  PURPURA  H^EMORRHAGICA  OF  BABES. 

Obtained  by  Babes  (1890)  from  the  spleen  and  lungs  of  an  individual  who 
died  from  purpura  haemorrhagica  with  symptoms  of  septicaemia.  Resembles 
the  bacillus  previously  described  by  Tizzoni  and  Giovannini,  and  still  more 
that  of  Kolb ;  but,  according  to  Babes,  differs  in  some  respects  from  both  of 
these,  although  they  all  belong  evidently  to  the  same  group. 

Morphology. — Bacilli  with  rounded  ends,  oval  or  pear-shaped,  about  0.3  p 
thick,  surrounded  by  a  narrow  capsule. 

Stains  with  the  aniline  colors,  but  not  deeply,  and  still  less  intensely  by 
Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  In  gelatin  stick  cultures,  at  the 
end  of  three  days,  a  thin,  transparent,  irregular  layer  has  developed  upon 
the  surface,  and  a  whitish,  punctate  stripe  along  the  line  of  inoculation.  In 
agar  stick  cultures  an  abundant  development  occurs  along  the  line  of  punc- 
ture, and  at  the  end  of  three  days  the  growth  upon  the  surface  consists  of 
small,  moist,  transparent  drops;  later  of  larger,  flat,  shining,  yellowish- 
white  plaques  which  have  ill-defined  margins.  Upon  blood  serum  the  de- 
velopment is  somewhat  more  abundant  in  the  form  of  small,  white,  moist 
colonies  one  to  two  millimetres  broad.  Upon  potato,  at  the  end  of  three 
days,  moist,  whitish  drops  with  ill-defined  margins. 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  511 

Pathogenesis. — Inoculations  in  the  conjunctivas  of  rabbits  produce  ecchy- 
moses  of  the  conjunctiva.  At  the  autopsy  numerous  hasmorrhagic  extrava- 
sations are  found  in  all  the  organs,  especially  in  the  lungs  and  liver;  the 
spleen  is  enlarged ;  the  bacilli  can  be  recovered  in  pure  cultures  from  the 
various  organs.  Old  cultures  proved  to  have  lost  their  virulence.  Patho- 
genic for  mice,  which  die  from  general  infection  in  the  course  of  a  few  days ; 
the  spleen  is  enlarged,  and  haemorrhages  in  the  serous  membranes  are  usually 
seen. 

147.    BACILLUS  OF  PURPURA  H^MORRHAGICA  OF  KOLB. 

Obtained  by  Kolb  (1891)  from  the  various  organs  of  three  individuals 
who  died  in  from  two  to  four  days  from  attacks  characterized  by  suddenly 
developed  feveV,  purpura,  and  albuminous  urine. 

Morphology. — Oval  bacilli,  usually  in  pairs,  0.8  to  l.Syulong  andO.8/* 
broad,  surrounded  by  a  narrow  capsule,  which  is  only  seen  distinctly  in 
preparations  from  the  organs. 

Stains  with  the  aniline,  colors,  but  not  deeply,  and  still  more  feebly  by 
Gram's  method. 

Biological  Characters.  — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  non-motile  bacillus.  Does  not  form  spores.  Grows  iri  the  usual 
culture  media  at  the  room  temperature.  In  gelatin  stick  cultures,  at  the  end 
of  four  days,  a  very  small,  thin,  hyaline  growth  is  seen  about  the  point  of 
inoculation.  The  development  is  more  abundant  along  the  line  of  puncture. 
Upon  the  surface  of  agar  a  thin  layer  is  formed  with  smooth  margins. 
Upon  potato,  at  the  end  of  three  to  four  days,  a  whitish,  moist,  shining  stripe 
is  seen  along  the  impfstrich  which  is  about  three  millimetres  broad. 

Pathogenesis. — Injections  of  0.5  to  1  cubic  centimetre  of  a  bouillon 
culture  into  the  abdominal  cavity  of  rabbits  cause  symptoms  of  general  in- 
fection in  the  course  of  a  few  days,  and  not  infrequently  haemorrhagic  ex- 
travasations are  seen  in  the  ear  muscles.  More  than  one  cubic  centimetre 
may  cause  death  in  from  one  to  three  days.  At  the  autopsy  haemorrhagic 
extravasations  are  found  in  the  subcutaneous  tissues  and  in  the  serous  and 
mucous  membranes.  The  blood  has  little  disposition  to  coagulate;  the 
bacillus  may  be  recovered  in  pure  cultures  from  the  various  organs.  In 
guinea-pigs  local  ecchymoses  are  sometimes  produced,  otherwise  not  natho- 
geiiic  for  this  animal.  Pathogenic  for  mice,  which  die  from  general  infec- 
tion, after  being  inoculated  with  a  small  quantity  of  a  pure  culture,  in  from 
two  to  three  days;  spleen  enlarged;  lymphatic  glands  often  haemorrhagic. 
Not  fatal  to  dogs,  but  animals  which  were  inoculated  with  one  cubic  centi- 
metre of  a  bouillon  culture  and  subsequently  killed  proved  to  have  haemor- 
rhagic  extravasations  in  the  various  organs. 

148.    BACILLUS   HEMINECROBIOPHILUS. 

Obtained  by  Arloing  (1889)  from  a  caseous  lymphatic  gland  in  a  guinea-pig. 

Morphology. — Bacilli  which  vary  greatly  in  length  and  are  sometimes  so 
short  as  to  resemble  micrococci  ("polymorphous");  usually  from  one  to 
four  n  long;  in  anaerobic  cultures  from  eight  to  twenty  #. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  slightly  motile  bacillus.  Spore  formation  not  observed.  Grows 
rapidly  in  the  usual  culture  media — best  in  the  incubating  oven  at  35°  C. 
The  growth  upon  the  surface  of  gelatin  has  a  yellowish  color.  Upon  potato 
a  yellowish-white  layer  is  developed. 

Pathogenesis. — According  to  Arloing,  this  bacillus  is  not  pathogenic  when 
injected  into  healthy  tissues  in  dogs,  sheep,  guinea-pigs,  and  rabbits,  but 
when  the  tissues  have  previously  been  injured  it  produces  a  local  oedema  and 
necrotic  changes,  accompanied  by  gas  formation.  This  is  not  peculiar  to  the 
microorganism  described  by  Arloing,  which  appears  to  be  one  of  the  Proteus 
group. 


512  PATHOGENIC   AEROBIC  BACILLI 

149.    STREPTOCOCCUS  CONGLOMERATE   (Kurth). 

Obtained  by  Kurth  (1890)  from  cases  of  scarlet  fever. 

Morphology. — As  obtained  from  bouillon  cultures  it  consists  of  masses 
made  up  of  chains  of  cocci;  free  chains  are  only  occasionally  seen. 

Biological  Characters. — This  streptococcus  is  said  to  differ  from  Strepto- 
coccus pyogenes  and  various  other  previously  described  streptococci  by  the 
fact  that  in  bouillon  cultures,  at  a  temperature  of  37°  C. ,  it  forms  at  the  bot- 
tom of  the  tube  smooth,  round,  and  very  firm  white  scales,  or  a  single  flat 
layer  which  is  not  disintegrated  when  the  tube  is  slightly  agitated ;  other 
streptococci  are  said  to  form  a  loose  deposit  which  is  either  entirely  broken 
up  or  forms  viscid  threads  when  the  tube  is  gently  rotated. 

Pathogenesis. — Very  pathogenic  for  mice.  * 

150.  BACILLUS  CAPSULATUS  Mucosus  (Fasching). 

Obtained  from  the  nasal  secretion  in  two  cases  of  influenza. 

Morphology.— Bacilli  from  3  to  4  /*  long  and  0.75  to  1  /*  thick,  enveloped 
in  a  capsule  containing  one  to  four  individuals. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile,  non-liquefying  bacillus.  Does  not  form  spores.  Grows  in  the  usual 
culture  media  at  18°  to  35°  C.  Upon  gelatin  plates  circular,  milk-white  colo- 
nies are  developed ;  these  have  a  faint  aromatic  odor  and  are  cupped  upon 
the  upper  surface ;  they  resemble  drops  of  mucus  about  the  size  of  a  pin's  head. 
In  stick  cultures  in  gelatin  a  nail-like  growth,  like  that  of  Friedlander's  bacil- 
lus, is  seen,  and  there  is  a  formation  of  gas. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Pathogenesis. — White  mice  and  field  mice  die  from  general  infection  in 
from  thirty-six  to  forty-eight  hours  after  inoculation ;  they  also  suffer  from 
conjunctivitis.  Not  pathogenic  for  rabbits  or  for  pigeons. 


151.   BACILLUS  PYOGENES  SOLI. 

Obtained  by  Bolton  from  garden  earth  by  inoculation  into  a  rat.  Found 
in  association  with  the  tetanus  bacillus  in  pus  from  the  inoculation  wound. 

Morphology. — Closely  resembles  the  bacillus  of  diphtheria.  "  It  presents 
the  same  irregularities  of  shape,  and  the  transverse,  unstained  clear  spares 
in  stained  preparations,  as  the  diphtheria  bacillus.  The  individual  bacilli 
vary  greatly  in  length  and  thickness,  and  many  of  them  are  bent  and  nar- 
rower through  the  middle  than  at  the  poles." 

Stains  readily  with  the  usual  aniline  colors,  but  takes  the  stain  irregularly, 
sometimes  showing  deeply  stained  spots  which  may  be  perfectly  round.  Does 
not  stain  by  Gram's  method. 

Biological  Characters.  —  An  aerobic  and  facultative  anaerobic,  non- 
Uquefying,  non-motile  bacillus.  Spore  formation  not  observed  with  cer- 
tainty —  highly  refractive  ovoid  bodies  are  sometimes  met  with,  but  these  do 
not  seem  to  be  specially  resistant  to  heat.  In  gelatin  roll  tubes  very  small, 
spherical  colonies  are  developed,  which  under  a  low  power  are  seen  to  !><• 
finely  granular  and  to  have  a  lemon-yellow  color.  Grows  best  in  a  slight  1  y 
acid  medium — very  slowly  at  the  room  temperature.  In  gelatin  stick  cul- 
tures isolated  colonies  are  formed  along  the  line  of  puncture.  Scanty  growt  h 
on  potato  or  blood  serum.  Bolton  says :  "  I  have  rarely  succeeded  in  getting 
a  growth  in  agar." 

Pathogenesis. — Subcutaneous  inoculations  in  rats,  gray  mice,  rabbits, 
and  usually  in  white  mice  produce  an  abscess  at  the  point  of  inoculation. 
Injections  into  the  ear  veins  of  rabbits  sometimes  give  rise  to  multiple  ab- 
scesses, especially  in  the  joints  and  kidneys.  "  The  abscesses  following  sub- 
cutaneous inoculation  form  very  quickly,  within  twenty-four  hours,  and  run 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  513 

a  longer  or  shorter  course,  from  forty -eight  hours  to  eight  or  ten  days,  in 
direct  proportion  to  the  amount  of  the  culture  introduced.  The  animals  do 
not  seem  to  suffer  any  inconvenience,  as  a  rule,  and  after  the  abscess  is 
opened  suppuration  ceases.  The  organism  is  found  aggregated  in  small  and 
large,  irregular  clumps  in  the  pus,  many  of  them  lying  in  the  pus  corpuscles. 
It  seems  to  form  metastatic  abscesses  only  under  exceptional  circumstances, 
such  as  when  injected  directly  into  the  blood.  Otherwise  the  abscess  remains 
strictly  confined  to  the  seat  of  inoculation  in  rabbits,  white  rats,  and  gray 
mice." 

152.   BACILLUS  VENENOSUS. 

Obtained  by  Vaughan  from  water. 

Morphology. — Bacilli  with  rounded  ends,  two  to  four  times  as  long  as 
broad. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  actively  motile  bacillus.  Spore  formation  not  mentioned.  Grows 
rapidly  in  the  usual  culture  media  at  the  room  temperature — also  at  38°  C. 
On  gelatin  plates  small,  white,  spherical  colonies — sometimes  slightly  yel- 
low; the  superficial  colonies  are  elevated  above  the  surface  of  the  gelatin. 
In  gelatin  tubes  an  abundant  growth  occurs  along  the  line  of  puncture  and 
slowly  extends  upon  the  surface.  In  cultures  from  the  spleen  of  an  inocu- 
lated animal  the  growth  upon  the  surface  is  less  marked.  On  agar  a  thin, 
white  layer  is  formed.  On  potato  a  light-brown,  moist  growth.  In  recent 
cultures  from  the  spleen  of  an  inoculated  animal  the  growth  upon  potato 
may  be  invisible.  Grows  abundantly  both  in  Parietti's  solution  and  in  Uf- 
felmann's  gelatin. 

Pathogenesis.  —Pathogenic  for  rats,  mice,  guinea-pigs,  and  rabbits. 

153.    BACILLUS  VENENOSUS  BREVIS. 

Obtained  by  Vaughan  from  water. 

Morphology. — Short,  thick  bacilli,  about  twice  as  long  as  broad;  in  old 
cultures  grows  out  into  threads. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  actively  motile  bacillus.  Spore  formation  not  mentioned. 
Grows  rapidly  in  the  usual  culture  media  at  the  room  temperature— also  at 
38°  C.  On  gelatin  plates  forms  small,  round  colonies  with  concentric  rings; 
the  deeper  colonies  are  generally  yellowish  or  brown ;  the  surface  colonies 
are  elevated  and  spread  but  little.  In  gelatin  tubes  grows  along  the  line  of 
puncture  and  spreads  slowly  upon  the  surface,  finally  reaching  the  sides  of 
the  tube.  Upon  agar  a  thin,  white  layer  is  formed.  On  potato  a  thick  and 
moist,  light-brown  growth.  When  kept  for  fourteen  days  or  longer  at  40°  C. 
there  is  an  invisible  growth  upon  potato.  Grows  abundantly  in  Parietti's 
solution  and  slowly  in  Uffelmann's  gelatin. 

Pathogenesis. — Pathogenic  for  rats,  mice,  guinea-pigs,  and  rabbits. 

154.    BACILLUS   VENENOSUS  INVISIBILIS. 

Obtained  by  Vaughan  from  water. 

Morphology. — A  slender  bacillus  with  rounded  ends,  from  two  to  four 
times  as  long  as  broad. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Spore  formation  not  mentioned.  Grows  slowly 
in  the  usual  culture  media  at  the  room  temperature — also  at  38°  C.  On  gela- 
tin plates  small,  granular,  yellowish  colonies  are  developed ;  the  superficial 
colonies  are  coarsely  granular  and  very  irregular  in  size  and  outline.  In 
gelatin  tubes  grows  slowly  both  on  the  surface  and  along  the  line  of  punc- 
ture ;  scarcely  visible  at  end  of  three  days.  On  agar  a  very  thin,  white 
3« 


514  PATHOGENIC   AEROBIC   BACILLI 

growth.  On  potato  the  growth  is  sometimes  invisible ;  on  some  potatoes  a 
light-brown  layer  may  be  developed.  Grows  well  both  in  Parietti's  solution 
and  in  Uffelmann's  gelatin. 

Pathogenesis. — Pathogenic,  but  in  less  degree  than  Bacillus  venenosus. 

155.    BACILLUS   VENENOSUS   LIQUEFACIENS. 

Obtained  by  Vaughan  from  water. 

Morphology. — Bacilli  with  rounded  ends,  one  and  one-half  to  twice  as 
long  as  broad. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  lique- 
fying, motile  bacillus.  Spore  formation  not  mentioned.  Grows  rapidly  in 
the  usual  culture  media  at  the  room  temperature— also  at  38°  C.  On  gehttni 
plates  the  deep  colonies  are  finely  granular,  spherical,  and  yellowish  iu 
color;  superficial  colonies  elevated  and  spread  over  the  surface.  In  gelatin 
tubes  grows  abundantly  along  the  line  of  puncture  and  spreads  slowly  over 
the  surface;  liquefaction  commences  in  from  four  to  six  weeks.  On  agar  a 
thin,  white  growth.  On  potato  a  moist,  light-brown  or  yellowish  growth. 
When  kept  for  fourteen  days  or  longer  on  spleen  tissue  it  forms  an  invisible 
growth  on  potato.  Grows  abundantly  both  in  Parietti's  solution  and  in 
Uffelmann's  gelatin. 

Pathogenesis. — Pathogenic  for  mice,  rats,  guinea-pigs,  and  rabbits. 

156.    BACILLUS  AEROGENES   CAPSULATUS. 

Found  by  Welch  in  the  blood  vessels  of  a  patient  with  thoracic  aneurism 
opening  externally ;  autopsy  made  in  cool  weather  eight  hours  after  death — 
the  vessels  found  full  of  gas  bubbles. 

Morphology. — Straight  or  slightly  curved  bacilli  with  slightly  rounded 
or  sometimes  square-cut  ends ;  a  little  thicker  than  Bacillus  aiithracis,  and 
varying  in  length — average  length  3  to  6  fj. ;  long  threads  and  chains  are  oc- 
casionally seen.  The  bacilli,  both  from  cultures  and  in  the  animal  body,  are 
enclosed  in  a  transparent  capsule. 

Biological  Characters. — An  anaerobic,  non-motile,  non-liquefying  ba- 
cillus. Does  not  form  spores.  Grows  in  the  usual  culture  media,  in  the  ab- 
sence of  oxygen,  at  the  room  temperature,  and  produces  an  abundant  de- 
velopment of  gas  in  all.  In  nutrient  gelatin  there  is  no  marked  liquefaction, 
but  the  gelatin  is  slightly  peptonized.  In  agar,  colonies  are  developed  which 
are  usually  one  to  two  millimetres  in  diameter,  but  may  attain  a  diameter  of 
one  centimetre ;  they  are  grayish- white  in  color  and  in  the  form  of  flattened 
spheres,  ovals,  or  irregular  masses,  beset  with  little  projections  or  hair-like 
processes.  Bouillon  is  rendered  diffusely  cloudy,  with  an  abundant  white 
sediment.  Milk  is  coagulated  in  one  or  two  days.  The  cultures  in  agar  and 
bouillon  have  a  faint  odor,  comparable  to  that  of  stale  glue.  Upon  potato  & 
pale  grayish-white  layer  is  developed;  growth  occurs  at  18°  to  20°  C.,  but  is 
much  more  rapid  at  30°  to  37°  C.  Bouillon  cultures  are  sterilized  by  ex- 
posure to  a  temperature  of  58°  C.  for  ten  minutes. 

Pathogenesis. — "Quantities  up  to  2.5  cubic  centimetres  of  fresh  bouillon 
cultures  were  injected  into  the  circulation  of  rabbits  without  an y  apparent 
effect,  except  in  one  instance  in  which  a  pregnant  rabbit  was  killed,  by  the 
injection  or  one  cubic  centimetre,  in  twenty -one  hours.  If  the  animal  is 
killed  shortly  after  the  iniection  the  bacilli  develop  rapidly  after  death,  with 
an  abundant  formation  of  gas  in  the  blood  vessels  and  organs,  especially  t  lie 
liver.  At  temperatures  of  18°  to  20J  C.  the  vessels,  organs,  and  serous  cavi- 
ties may  be  full  of  gas  in  eighteen  to  twenty-four  hours,  and  at  tempera- 
tures or  30°  to  32°  C.  in  four  to  six  hours,  when  one  cubic  centimetre  of  a 
bouillon  culture  has  been  injected  into  the  circulation  shortly  before  death." 

It  is  suggested  by  Welch  and  Nuttall  that  in  some  of  the  cases  in 
which  death  has  been  attributed  to  the  entrance  of  air  into  the  veins,  the  gas 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  515 

found  at  the  autopsy  may  not  have  been  atmospheric  air,  but  may  have  been 
produced  by  this  or  some  similar  microorganism  entering  the  circulation  and 
developing  after  death. 

157.    BACILLUS  OF  CANON  AND  PIELICKE. 

"Found  by  Canon  and  Pielicke  (1892)  in  the  blood  of  fourteen  patients 
with  measles,  and  supposed  to  be  the  etiological  agent  in  this  disease. 

Morphology. — Bacilli  varying  greatly  in  size;  sometimes  the  length  is 
equal  to  the  diameter  of  a  red  blood  corpuscle,  others  are  quite  short  and 
resemble  diplococci ;  often  united  in  pairs. 

Stained  by  Canon,  in  blood  drawn  from  the  finger,  by  the  use  of  the  fol- 
lowing solution :  Concentrated  aqueous  solution  of  methylene  blue,  forty 
cubic  centimetres ;  one-quarter-per-cent  solution  of  eosin  in  seventy-per-cent 
alcohol,  twenty  cubic  centimetres;  distilled  water,  forty  cubic  centimetres. 
The  preparations  were  first  placed  in  absolute  alcohol  for  five  to  ten  minutes, 
then  placed  in  the  staining  solution  in  the  incubating  oven  at  37°  C.  from 
six  to  twenty  hours.  Some  of  the  bacilli  do  not  stain  uniformly,  but  present 
the  appearance  of  stained  spots  alternating  with  unstained  portions. 

Biological  Characters  not  determined.  Does  not  grow  in  glycerin-agar 
or  in  blood  serum.  In  bouillon  inoculated  with  blood  from  the  finger  of  a 
measles  patient,  bacilli  were  obtained  in  three  cultures  which  resembled  the 
bacillus  found  in  the  blood,  and  which  failed  to  grow  when  transplanted  to- 
glycerin-agar,  blood  serum,  or  bouillon.  At  first  the  bouillon  remained 
clear,  with  a  sediment  at  the  bottom  partly  made  up  of  the  inoculated  blood ; 
after  several  days  a  faint  cloudiness  was  noticed  and  small  flocculi  formed. 
In  these  bouillon  cultures  the  bacilli  had  various  forms  and  dimensions, 
some  of  them  exceeding  in  length  those  found  in  stained  preparations  from 
the  blood.  They  appeared  to  have  a  slight  independent  motion.  The  bacilli 
in  these  bouillon  cultures  did  not  stain  by  Gram's  method.  The  bacilli  re- 
ferred to  were  found  in  the  blood  preparations  in  varying  numbers — some- 
times very  few,  and  at  others  the  first  field  examined  was  crowded.  They 
were  found  during  the  whole  course  of  the  disease,  and  in  one  case  three 
days  after  the  fever  had  disappeared.  They  were  also  found  in  the  secre- 
tions from  the  nose  and  conjunctiva  of  measles  patients. 

158.    BACILLUS   SANGUINIS    TYPHI. 

Obtained  (1892)  by  Brannan  and  Cheesman  from  the  blood  of  typhus- 
fever  patients.  "The  blood,  obtained  under  strict  antiseptic  precautions 
from  the  six  living  patients,  was  streaked  on  six-per-cent  glycerin-agar 
plates,  and  smeared  on  sterilized  cover  glasses  by  Dr.  Brannan  and  brought 
at  once  to  the  laboratory.  The  cover-glass  smears  from  all  the  cases,  being' 
dried  at  once  in  the  air,  were  fixed  in  alcohol  and  stained  in  Czenzynski's 
solution  for  eighteen  hours  at  room  temperature.  Although  all  of  these 
covers  were  examined  throughout  with  a  one-sixteenth  homogeneous  immer- 
sion lens  in  the  most  careful  manner,  in  only  about  one-half  of  them  a  few 
blue-stained  bacilli  were  found,  never  more  than  eight  or  ten  on  a  cover." 

Morphology. — Bacilli  with  round  ends,  from  1  to  2. 5  ju  long  and  0.5  to 
0.8  u  broad  ;  solitary  or  in  pairs,  and  occasionally  in  chains  containing  six 
to  eight  elements;  often  club-shaped,  or  ovoid  in  recent  cultures. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile  bacillus.  Does  not  form  spores.  Does  not  grow  at  a  lower  tempera- 
ture than  27°  C.  Grows  best  upon  blood  serum  at  37.5°  C.  Upon  glycerin- 
agar  plates  colonies  are  developed  which  at  the  end  of  eighteen  hours  appear 
as  minute,  bluish-gray,  translucent  spots,  the  diameter  of  which  does  not 
exceed  0.25  millimetre  ;  later  the  colonies  appear  dry  and  scaly,  they 
are  flat,  more  opaque,  and  whiter,  and  do  not  exceed  two  millimetres  in 


510 


PATHOGENIC   AEROBIC  BACILLI 


diameter.  Under  a  low  power  the  recent  colonies  are  seen  to  be  granular, 
to  have  a  sinuous  and  sharply  defined  margin  and  a  pale-brown  color  which 
is  more  intense  at  the  centre  and  in  scattered  points  upon  the  surface.  When 
magnified  one  hundred  diameters  the  surface  appears  to  be  coarsely  granular, 
and  coarse,  irregular  spiculae  are  seen  about  the  margin.  In  glycerin-agar 
tubes,  at  37. 5°  C.,  growth  occurs  upon  the  surface  and  along  the  line  of 
puncture  as  small,  white,  isolated  colonies.  Upon  blood  serum  a  slightly 
elevated,  white,  shining  layer  is  developed.  In  milk  a  white  deposit  is 
formed  at  the  bottom  of  the  tube  and  the  milk  undergoes  no  apparent  change. 
On  potato  no  visible  growth  was  obtained. 

Pathogenesis.— "Inoculations  of  cultures  of  the  bacillus  obtained  from 
two  of  the  cases  were  made  in  eight  rabbits,  two  guinea-pigs,  and  two  white 
mice.  All  the  animals  showed  marked  emaciation,  and,  with  the  exception 
ot  two  rabbits,  all  the  animals  experimented  upon  died  in  from  ten  to  twenty- 
nine  days.  The  inoculated  bacillus  was  obtained  from  the  heart's  blood  of 
two  of  the  rabbits  that  died." 


Fia.  159.  FlO.  160. 

FIG.  159.— Bacillus  gracilis  cadaveris,  from  a  gelatin  culture.  X  1,000.  From  a  photomicro- 
graph. (Sternberg.) 

FIG.  160.— Bacillus  gracilis;  colonies  in  gelatin  roll  tube,  end  of  forty-eight  hours.  X  12.  From 
a,  photograph.  (Sternberg  ) 

159.  BACILLUS  GRACILIS  CADAYERIS  (Sternberg). 

Obtained  (1889)  from  a  fragment  of  liver,  of  man,  kept  for  forty-eight 
hours  in  an  antiseptic  wrapping. 

Morphology. — Bacilli  about  1  //  broad  and  2  ft  long,  associated  in  long 
chains. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile,  non-liquefying  bacillus.  Spore  formation  not  observed.  In  gelatin 
roll-tubes  the  deep  colonies  are  opaque  and  spherical ;  superficial  colonies 
circular  or  slightly  irregular  in  outline,  white  in  color,  and  opaque  or  slightly 
translucent.  In  gelatin  stick  cultures,  at  22°  C.,  at  the  end  of  fiv<>  days  a 
rather  thick,  white  mass  at  tho  point  of  puncture,  covering  one-third  of  tin- 
sin-face,  and  closely  crowded,  opaque  colonies  at  bottom  of  line  of  puncturr. 
with  slander,  branching  outgrow! li  above.  In  nutrient  agar,  at  the  end  of 
five  days  at  22°  C.,  a  milk-white  growth  upon  the  surface  and  opaque 
growth  to  bottom  of  line  of  puncture.  On  potato,  at  end  of  five  days  at 
22°  C.,  rather  thick,  cream-white  growth  witn  irregular  margins  along  the 
impfstrich.  Cultures  in  bouillon  have  a  milky  opacity  and  a  very  disagree- 
able odor.  Grows  in  agna  coco  without  formation  of  <ras. 

Pathogenic  for  rabbits  when  injected  into  the  cavity  of  the  abdomen. 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  517 

160.    CAPSULE   BACILLUS   OF   CHIARI. 

Obtained  by  Chiari  (1895)  from  a  man  who  died  from  an  ascending  ne- 
phritis, with  endocarditis  and  finally  meningitis. 

Morphology.—  Bacilli  about  2 p thick  and  from  3  to  4 //long,  some  as  long 
as  8  //,  with  rounded  ends,  and  surrounded  by  a  capsule. 

Biological  Characters.—  Similar  to  those  of  Friedlander's  bacillus,  from 
which  it  is  differentiated  by  the  following  characters :  Cocci-like  forms  less 
numerous;  growth  on  blood  serum  less  vigorous;  growth  in  bouillon  not 
so  abundant ;  very  pathogenic  for  rabbits  when  injected  into  the  circulation, 
and  to  mice  when  injected  subcutaneousiy.  (Probably  a  pathogenic  variety 
of  Friedlander's  bacillus.— G.  M.  S.) 

161.    BACILLUS   OF   HARRIS. 

Obtained  by  Harris  (1892)  from  an  abscess  wall  in  a  case  in  which  death, 
occurred  from  a  cerebral  abscess  consecutive  to  otitis  media. 

Morphology. — Described  by  Harris  as  "a  diplococcus  en  capsuled,  grow- 
ing into  paired  rods  and  chains  of  rods,  encapsulated  in  tissues."  Rods  are 
from  4 to  6//  long  and  1  //broad. 

Biological  Characters. — An  aerobic,  non-liquefying  bacillus.  The  Lis- 
ton  gold  medal  of  University  College,  London,  was  awarded  to  Harris  for 
his  paper  relating  to  this  bacillus,  which  he  describes  as  "a  new  microorgan- 
ism of  spreading  oedema."  Notwithstanding  this  fact,  his  account  of  his 
new  bacillus  is  very  incomplete.  He  describes  it  as  follows: 

Potato. — It  grows  rather  slowly  at  16°  C.,  in  softish,  moist,  dotted  colo- 
nies, which  later  coalesce,  forming  a  thinnish  layer  on  the  potato.  The  color 
is  somewhat  buff,  but  after  repeated  cultivation  tends  to  fade  until  a  cream- 
colored  culture  is  obtained.  The  surface,  though  rather  moist,  is  rough  and 
irregular.  The  growth  is  never  extensive  or  thick. 

Agar. — It  grows  in  a  thin  layer,  with  a  tendency  to  spread.  The  thick- 
ness of  the  growth  is  unequal  and  the  surface  uneven,  though  tending  to  be 
moist.  The  color  is  cream  in  the  less  transparent  portions  of  the  growth. 
Occasionally,  at  certain  points,  there  is  a  tendency  to  an  indipping  of  the 
growth. 

Gelatin. — It  does  not  liquefy.  Same  appearance  as  on  agar.  The  edge 
of  the  colony  under  a  low  power  is  seen  to  be  irregularly  rounded  and  fis- 
sured. The  growth  seems  to  proceed  radially  from  many  points  in  the 
same  neighborhood,  and  hence  the  rounded  and  lobed  appearance,  not  un- 
like a  thick  section  of  pancreas.  The  naked-eye  irregularities  on  the  surface 
are  evidently  due  to  this  mode  of  growth.  Under  the  microscope  there  is, 
in  the  thicker  portions,  a  brownish  tinge. 

Broth. — There  is  some  clouding,  and  a  white,  sandy  deposit  is  seen  after 
twenty-four  hours,  at  a  temperature  of  34°  C. 

162.    CAPSULE   BACILLUS   OF  NICOLAIER. 

Obtained  by  Nicolaier  (1894)  from  pus  contained  in  an  abscess  of  the  kid- 
ney— obtained  post-mortem. 

Morphology. — Thick  bacilli,  with  rounded  ends,  usually  four  times  as 
long  as  thick,  and  frequently  presenting  irregular  outlines  ;  often  united  in 
pairs,  and  sometimes  growing  out  into  filaments;  cocci-like  forms  also  occur. 
Often  surrounded  by  a  capsule  which  remains  unstained  in  stained  prepara- 
tions. Does  not  stain  by  Gram's  method. 

Biological  Characters. — An  aerobic,  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  Grows  at  the  room 
temperature  and  more  rapidly  at  37°  C.  Upon  gelatin  plates  at  20°  C.,  at 
the  end  of  twenty-four  to  thirty-six  hours  punctiform  colonies  are  devel- 
oped, which  under  a  low  power  appear  finely  granular,  and  grayish-yellow 


518  PATHOGENIC  AEROBIC  BACILLI 

spheres.  At  the  end  of  forty-eight  to  sixty  hours  the  superficial  colonies  ap- 
pear as  round  or  slightly  irregular,  grayish- white  discs,  which  project  but  lit- 
tle above  the  surface  of  the  gelatin,  and  have  thin,  transparent  margins. 
The  deep  colonies  have  a  sharply  denned  contour,  with  dark-brown  centre 
and  a  purely  granular  pale-brown  marginal  zone.  In  gelatin  stick  cult 
a  slightly  elevated,  moist-looking,  sticky  layer  with  more  or  less  transparent 
margins  is  developed.  In  slanting  cultures  this  growth  gradually  slips  down 
to  the  lowest  part  of  the  exposed  surface,  leaving  a  thin,  gray,  transparent 
layer  over  the  gelatin ;  along  the  line  of  puncture  a  ribbon-like,  grayish - 
wnite  growth  with  irregular  margins  is  developed.  In  media  containing 
glucose  some  gas  bubbles  are  developed.  The  growth  is  much  more  rapid 
in  the  incubating  oven  at  37°  C.,  and  there  is  an  abundant  development  of 
gas  in  agar  tubes.  Upon  potato  a  grayish- white,  slimy  mass  with  a  shining 
surface  is  quickly  developed.  In  bouillon,  at  the  end  of  twenty-four  hours, 
at  373  C.,  the  medium  is  clouded  throughout,  and  a  grayish- white  deposit  ac- 
cumulates at  the  bottom  of  the  tube.  Development  occurs  also  in  acid 
media. 

Pathogenesis. — Pathogenic  for  house  mice,  white  mice,  and  for  rats — not 
for  rabbits  or  guinea-pigs — by  subcutaneous  injections.  As  Nicolaier  lias 
made  a  careful  comparison  of  the  characters  of  the  various  "  capsule  bacilli" 
described,  we  quote  from  him  as  follows  : 

"Our  bacillus  in  its  morphologv  and  growth  in  various  media  closely  re- 
sembles that  of  Fasching  and  of  Abel,  both  of  which  were  obtained  in  patho- 
logical products  from  man.  It  is  distinguished  from  them  by  its  pathogenic 
action  upon  mice.  "White  and  gray  mice  when  infected  with  our  bacillus 
die  from  septicaemia  and  show,  in  addition  to  a  serous  exudation  at  the  point 
of  inoculation,  constant  pathological  changes  in  the  kidneys,  which  may  usu- 
ally be  recognized  by  a  macroscopic  examination.  Also  by  the  spleen,  which 
is  mot  always  enlarged,  and  the  liver,  which  only  in  a  few  cases  showed  any 
microscopic  changes.  In  mice  inoculated  with  the  bacillus  of  Fasching,  or 
that  of  Abel,  which  died  of  septicaemia,  there  was  constantly  seen  an  en- 
largement of  the  spleen  (Fasching,  Abel)  and  of  the  liver  (Abel),  and  a 
cloudy  swelling  of  the  liver  and  kidneys  (Abel)  which  our  mice  failed  to 
show.  The  macroscopic  and  microscopic  changes  which  we  found  in  the 
kidnevs  in  mice,  and  also  in  some  cases  in  the  liver  and  spleen,  were  not  ob- 
served by  Fasching  or  by  Abel.  Recently  Paulsen  has  described  a  capsule  ba- 
cillus from  atrophic  rhinitis,  and  Marchand  a  capsule  bacillus — not  further 
described — which  he  obtained  in  great  numbers  from  the  exudate  in  a  case 
of  lobar  pneumonia.  Both  appear  to  be  very  similar  to  Fasching's  bacillus. 
They  are  pathogenic  for  mice,  but  do  not  cause  the  changes  in  the  kidneys 
which  we  have  described.  These  capsule  bacilli  are  therefore  not  i dent ieal 
with  ours.  Marchand\s  bacillus  is  further  distinguished  by  the  fact  that  it  is 
pathogenic  for  guinea-pigs.  .  .  .  The  bacillus  of  Kockel  is  distinguished 
from  ours  by  the  following  characters  :  It  forms  upon  the  surface  of  gelatin, 
as  well  as  in  stick  cultures,  highly  elevated,  button-like  colonies,  while  our 
bacillus  grows  more  in  flat  and  broad  layers.  It  also  lacks  the  semi-fluid 
character  of  growth  upon  slanting  agar,  which  distinguishes  our  bacillus, 
and  as  a  result  of  which  the  growth  slips  down  to  the  lowest  point  on  the 
slanting  surface  ;  further  it  forms  upon  potato  a  yellowish  layer,  while  ours 
is  grayish-white  ;  and  it  does  not  grow  in  acid  media.  Finally,  it  is  patho- 
genic for  rabbits  by  intravenous  injection,  while  ours  is  not." 

103.    BACILLUS   MUCOSUS   OZ^EN^l. 

Obtained  by  Abel  (1893)  from  cases  of  o/:«-n;i  simplex  (rhinitis  atrophicans 
fcetida).  As  this  bacillus  appears  to  correspond  in  its  morphological  and  bio- 
logical characters  with  the  capsule  bacillus  a  hove  described  (No.  162)  we 
shall  not  repeat  this  dcsei-iption.  but  quote  from  Abel,  as  follows  : 

"This  bacillus,  found  in  the  secretion  from  cases  of  ozama,  as  the  de- 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  519 

scription  we  have  given  shows,  closely  resembles  Friedlander's  pneumo- 
bacillus.  It  is  distinguished  from  it  by  certain  constant  characters.  The 
ozsena  bacillus  forms  in  cultures  a  more  fluid  mass  than  Friedlander's.  As 
a  result  of  this  it  does  not  form  the  characteristic  nail-head  culture,  but 
spreads  out  over  the  surface  of  the  gelatin.  Upon  slanting  gelatin  cultures 
the  growth  slips  down  to  the  lowest  point.  In  old  cultures  it  never  shows  a 
brown  coloring  of  the  culture  medium.  It  never  forms  gas  on  potato,  and 
in  agar  and  gelatin  cultures  but  little  gas  is  developed.  Mice  always  suc- 
cumb to  subcutaneous  inoculations,  while  Friedlander's  bacillus  does  not 
kill  mice.  Intraperitoneal  infection  of  guinea-pigs  with  the  ozaeiia  bacillus 
always  causes  their  death.  Friedlander's  bacillus  only  killed  about  half  the 
guinea-pigs  inoculated  in  the  cavity  of  the  abdomen.  Finally,  Friedlander's 
bacillus  has  a  greater  tendency  to  cocci-like  forms.  The  resemblance  to 
Pfeiffer's  capsule  bacillus  is  closer.  But  the  tenacious  layer  described  by 
Pfeiffer  as  found  upon  the  intestinal  coils  and  the  lungs  in  mice,  and  the 
sticky  condition  of  the  blood  and  tissue  juices  (fadenziehende)  are  want- 
ing. The  reaction  at  the  point  of  inoculation  in  mice  is  also  much  more 
pronounced  with  my  bacillus." 

It  seems  extremely  probable  that  this  bacillus,  the  Bacillus  capsulatus  mu- 
cosus  of  Fasching,  and  the  above-described  capsule  bacillus  of  Nicolaier 
are  simply  pathogenic  varieties  of  one  and  the  same  bacillus. 

164.    CAPSULE  BACILLUS   OF  VON  DUNGERN. 

Obtained  by  von  Dungern  (1893),  post  mortem,  from  a  new-born  child 
which  died  of  hemorrhagic  septicaemia — infection  through  umbilicus. 

Morphology. — A  short,  thick  bacillus,  from  1  to  2  /«  long  and  half  as 
broad,  surrounded  by  a  capsule  which  is  slightly  stained  by  gentian  violet — 
best  seen  in  the  body  of  infected  mice  ;  sometimes  seen  in  pairs  or  in  chains 
of  four  elements  ;  also  grows  out  into  filaments,  especially  in  bouillon. 
Upon  potato  usually  only  small  spherical  elements,  resembling  micrococci, 
are  seen.  Does  not  stain  by  Gram's  method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
motile,  non-liquefying  bacillus.  Does  not  form  spores.  Coagulates  milk, 
in  which  it  causes  an  abundant  development  of  gas  at  38°  C.  Has  feeble 
indol  reaction.  Grows  well  at  room  temperature,  more  rapidly  in  incubator. 
Upon  gelatin  plates  the  deep  colonies  at  end  of  twelve  hours  are  the  size  of  a 
pin's  head,  finely  granular,  spherical,  and  sharply  defined.  Upon  the  sur- 
face, porcelain-like,  elevated,  white  colonies  are  developed,  which  in  two  or 
three  days  attain  the  size  of  lentils.  In  gelatin  stick  cultures  development 
occurs  all  along  the  line  of  puncture,  frequently  with  formation  of  gas  bub- 
bles. Upon  agar  a  thick,  soft  layer  of  a  white  color  is  developed.  In  bouil- 
lon, at  38°  C.,  there  is  considerable  development  of  gas.  Upon  potato  the 
growth  is  very  abundant,  of  a  pale  yellowish- white  color,  thick,  soft,  some- 
what sticky,  and  filled  with  gas  bubbles.  A  great  portion  of  the  surface  is 
covered  by  this  growth  at  the  end  of  twenty -four  hours,  even  at  the  room 
temperature.  These  cultures  give  off  a  peculiar  odor,  sometimes  aromatic- 
foetid  and  sometimes  recalling  that  of  fresh  bread.  Some  of  the  cultures  on 
potato  soon  become  cream-like  in  consistence.  At  first  they  have  an  alkaline 
and  later  an  acid  reaction,  when  they  have  the  odor  of  acetic  acid. 

Pathogenesis. — Very  pathogenic  for  white  mice.  The  bacilli  are  found 
in  the  blood  and  in  all  the  organs  in  enormous  numbers.  At  the  point  of 
inoculation  there  is  frequently  a  hemorrhagic  oedema.  The  spleen  is  greatly 
enlarged.  Also  pathogenic  for  guinea-pigs  when  injected  into  the  cavity  of 
the  abdomen — less  pathogenic  for  rabbits. 

According  to  von  Dungern,  this  bacillus  can  not  be  distinguished  by  its 
morphological  and  biological  characters  from  Friedlander's  bacillus,  Bacil- 
lus capsulatus  of  Pfeiffer,  or  Bacillus  canalis  capsulatus  of  Mori.  But  it  is 
distinguished  from  these  by  greater  virulence,  especially  for  rabbits,  and  by 


520  PATHOGENIC  AEROBIC   BACILLI 

the  fact  that  it  frequently  gives  rise  to  hemorrhagic  extravasations  in  inocu- 
lated animals.  In  our  opinion  the  characters  given  do  not  justify  the  view 
that  this  bacillus  is  a  distinct  species  from  the  bacilli  above  mentioned. 

165.    BACILLUS  OF  BUNZL-FEDERN. 

Obtained  by  Bunzl-Federn  (1892)  from  the  sputum  of  a  patient  suffering 
from  pneumonia — by  inoculation  into  the  subcutaneous  tissues  of  a  rabbit. 

Morphology. — Bouillon  cultures  consist  mostly  of  "diplococci"  and 
short  bacilli,  but  upon  agar  slender  bacilli  and  long  filaments  are  commonly 
developed.  In  the  blood  of  infected  animals  it  also  varies  in  its  morphology. 
In  the  blood  of  rabbits  and  guinea-pigs  it  appears  as  short,  tolerably  thick 
rods,  which  frequently  show  polar  staining  and  resemble  diplococci.  In 
the  blood  of  white  mice  the  bacilli  are  of  the  same  thickness  but  longer — 
often  twice  as  long  as  in  rabbits.  Does  not  stain  by  Gram's  method  and  is 
without  a  capsule. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  In  gelatin  stick 
cultures  no  growth  is  observed  for  several  days.  At  the  end  of  eight  days  a 
thin,  grayish-white  layer  with  irregular  margins  is  developed,  and  discrete, 
punctiform,  grayish-white  colonies  are  seen  along  the  line  of  puncture.  In 
bouillon,  a  uniform  clouding  of  the  medium  occurs  within  twenty-four 
hours,  and  later  a  ring-formed  mycoderma  is  seen  upon  the  surface  around 
the  walls  of  the  test  tube.  After  some  days  the  bouillon  becomes  transparent 
and  a  slimy  deposit  remains  at  the  bottom,  which  is  coarsely  granular  <  >r 
lumpy. 

Upon  agar,  at  37°  C. ,  a  soft,  shining  layer  is  formed  at  the  end  of  twenty- 
four  hours ;  this  consists  of  fine  drops,  which  are  colorless  by  reflected  li<£ht 
and  grayish-white  bv  transmitted  light.  Usually  these  little  drops  do  not 
run  together.  In  milk,  development  occurs  without  coagulation  or  produc- 
tion of  acid.  Does  not  grow  on  potato. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  white  mice,  and  pig- 
eons. Subcutaneous  injections  of  0.4  to  1  cubic  centimetre  of  a  recent  bouil- 
lon culture  give  rise  to  septicaemia  and  death — in  rabbits  from  twelve  hours 
to  three  days,  in  guinea-pigs  in  from  two  to  four  days. 

166.   BACILLUS  OF  BUBONIC  PLAGUE   (Kitasato). 

Discovered  by  Kitasato  (1894)  in  the  blood  of  living  patients,  and 
in  the  buboes,  blood,  and  organs  of  those  who  had  recently  died  from 
the  infectious  malady  known  as  bubonic  plague.  Kitasato  was  sent 
to  Hong- Kong  by  the  Japanese  Government  for  the  purpose  of  inves- 
tigating this  disease.  According  to  Lowson  the  bacilli  are  found  in 
the  faeces,  in  the  contents  of  the  buboes,  and  in  the  blood. 

Morphology. — In  his  preliminary  note,  Kitasato  described  tin- 
plague  bacilli  as  "rods  with  rounded  ends,"  which  are  readily 
stained  by  the  ordinary  aniline  dyes,  the  poles  being  stained  darker 
than  the  middle  part,  especially  in  blood  preparations,  and  present- 
ing a  capsule  sometimes  well  marked,  sometimes  indistinct. 

Yersin,  who  was  sent  by  the  French  Government  to  study  the 
bubonic  plague  at  Hong-Kong,  arrived  in  that  city  on  the  15th  of 
June,  1894.  He  describes  the  bacillus  found  in  the  contents  of  the 
buboes  as  being  short  and  thick,  with  rounded  ends,  staining  easily 
with  the  aniline  colors,  but  not  by  Gram's  method.  "  The  extremities 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  521 

stain  more  intensely  than  the  centre,  so  that  they  often  present  a 
clear  space  in  the  middle.  Sometimes  the  bacilli  appear  to  be  sur- 
rounded by  a  capsule.  ...  In  bouillon  the  bacillus  has  a  very  char- 
acteristic appearance,  resembling  the  cultures  of  the  streptococcus  of 
erysipelas — a  clear  liquid  with  grumous  deposits  on  the  walls  and  at 
the  bottom  of  the  tube.  These  cultures  examined  under  the  micro- 
scope show  veritable  chains  of  short  bacilli,  presenting  in  places  a 
considerable  spherical  enlargement." 

Biological  Characters. — We  quote  from  Kitasato's  preliminary 
report  as  follows : 

The  bacilli  show  very  little  movement,  and  those  grown  in  the  incubator, 
in  beef -tea,  make  the  medium  somewhat  cloudy.  The  growth  of  the  bacilli  is 
strongest  on  blood  serum  at  the  normal  temperature  of  the  human  body 
(34°  C.) ;  under  these  conditions  they  develop  luxuriantly  and  form  a  col- 
ony moist  in  consistence  and  of  a  yellowish-gray  color ;  they  do  not  liquefy 
the  serum.  On  agar-agar  jelly  (the  best  is  good  glycerin  agar)  they  also 
grow  freely.  The  different  colonies  are  of  a  whitish-gray  color  and  by  re- 
flected light  have  a  bluish  appearance  ;  under  the  microscope  they  appear 
moist  and  in  rounded  patches  with  uneven  edges  ;  at  first  they  appear  every- 
where as  if  piled  up  with  "glass-wool,"  later  as  if  haying  dense,  large  cen- 
tres. If  a  cover-glass  preparation  is  made  from  a  cultivation  on  agar-agar, 
and,  after  haying  been  stained,  is  observed  under  the  microscope,  long 
threads  of  bacilli  are  seen,  which  might,  by  careless  inspection,  be  mistaken 
for  a  coccus  chain,  but  are  recognized  with  certainty  as  "  threads  of  bacilli " 
under  closer  observation.  The  growth  on  agar-gelatin  is  similar  to  that  on 
agar-agar  ;  in  a  puncture  cultivation  at  the  ordinary  temperature  after  a  few 
days  they  are  found  growing  as  a  fine  dust  in  little  points  alongside  the 
puncture,  but  with  very  little  growth  on  the  surface.  Whether  these  ba- 
cilli are  able  to  liquefy  ordinary  gelatin  or  not  I  am  at  present  unable  to  de- 
cide, as  the  temperature  of  Hong-Kong  ranges  so  high  that  the  employment 
of  simple  nutritive  gelatin  is  out  of  the  question.  I  shall  give  further  infor- 
mation on  this  question  later.  On  potatoes  at  a  temperature  of  from  28° 
to  30°  C.,  there  was  no  growth  after  ten  days'  observation,  but  at  a  tempera- 
ture of  37°  C.  the  bacilli  developed  sparingly  after  a  few  days  ;  the  growth 
was  whitish-gray  in  color  and  exsiccated.  As  mentioned  before,  the  bacilli 
grow  best  at  a  temperature  of  from  38°  to  39°  C. ;  at  how  low  a  temperature 
growth  is  possible  I  am  unable  at  present  to  state.  So  far  I  have  been  un- 
able to  observe  the  formation  of  spores. 

Experiments  on  Animals. — Mice,  rats,  guinea-pigs,  and  rabbits  are  sus- 
ceptible to  inoculation.  If  these  animals  are  inoculated  with  pure  culti- 
vations, or  with  the  blood  of  a  plague  patient  in  which  the  bacilli  have  been 
observed,  or  with  the  contents  of  a  bubo,  or  with  pieces  of  internal  organs, 
or  even  with  the  contents  of  the  intestine,  they  begin  to  become  ill  in  from 
one  to  two  days,  according  to  the  size  of  the  animal.  Their  eyes  become  wa- 
tery, they  begin  to  show  disinclination  for  any  effort,  later  they  avoid  their 
food,  and  hide  quietly  in  a  corner  of  the  cage.  The  temperature  rises  to 
41.5°  C.,  and  with  convulsive  symptoms  they  die  in  from  two  to  five  days.  I 
must  observe  that  in  Hong-Kong  I  could  only  obtain  small  guinea-pigs 
.  (weight  from  one  hundred  to  one  hundred  and  fifty  grammes)  and  small 
rabbits  (from  two  hundred  to  two  hundred  and  fifty  grammes).  If  I  could 
have  experimented  upon  larger  animals  it  is  possible  that  life  would  have 
been  prolonged  somewhat  beyond  the  periods  mentioned  above.  The  parts 
around  the  point  of  inoculation  are  infiltrated  with  a  reddish  gelatinous 
exudation,  the  spleen  is  enlarged,  sometimes  there  is  a  swelling  of  the  lym- 
phatic glands,  and  in  all  the  organs  the  bacilli  are  found.  The  results  found 


522  PATHOGENIC  AEROBIC  BACILLI 

after  death  in  animals  are  very  similar  to  those  found  in  anthrax  and  in 
oedema  malignum.  Pigeons  do  not  appear  to  be  susceptible  to  the  influence 
of  the  bacilli.  I  made  experiments  by  feeding  some  mice  and  guinea-pigs 
with  pure  cultivations  of  the  bacillus  and  with  small  pieces  of  the  internal 
organs  :  the  result  was,  such  animals  perished  in  a  few  days  under  the  same 
symptoms  as  those  which  had  been  inoculated.  In  all  the  internal  organs 
of  animals  so  destroyed  I  found  the  bacilli.  With  the  dust  of  dwelling- 
houses  from  which  the  plague-stricken  had  been  removed,  I  made  sev- 
eral experiments  upon  animals.  Some  of  the  animals  died  from  tetanus. 
In  one  case  only  a  guinea-pig  died  with  plague  symptoms,  and  in  this  ani- 
mal the  same  bacilli  were  found  in  the  internal  organs  as  in  those  of 
plague  patients  who  had  succumbed.  These  experiments  with  the  dust  from 
infected  houses  I  shall  certainly  continue.  Many  rats  and  mice  at  present 
die  spontaneously  in  Hong-Kong.  I  examined  some  of  them.  In  the  inter- 
nal organs  of  a  mouse  I  discovered  the  same  bacilli. 

Experiments  with  Desiccation, — The  contents  of  a  bubo  in  which  the 
bacilli  were  present  in  great  numbers  were  wiped  over  cover  glasses  (per- 
fectly cleansed  by  heat  and  alcohol),  and  some  of  these  cover-glasses  were 
dried  in  the  air  of  a  room  at  a  temperature  ranging  from  28°  to  30°  C.  Oth- 
ers I  exposed  directly  to  the  sun's  rays,  and  from  among  them,  after  an  expo- 
sure of  from  one,  two,  and  three  hours  up  to  six  days,  I  removed  some  parts. 
putting  such  portions  in  beef -tea  and  placing  them  in  the  incubator.  Those 
which  had  been  standing  in  the  room  from  one  to  thirty-six  hours  showed  a 
pretty  good  growth  in  the  incubator,  but  those  which  had  been  in  the  room 
for  more  than  four  days  were  unable  to  show  any  growth  even  after  one 
week's  incubation.  Those  exposed  directly  to  the  sun  were  all  destroyed  after 
from  three  to  four  hours.  Further  cultivations  on  serum  were  treated 
exactly  like  the  contents  of  the  bubo  with  very  similar  results. 

Experiments  with  Heat. — Beef -tea  cultivations  which  had  been  heated 
for  thirty  minutes  in  a  water  bath  up  to  80°  C.  were  destroyed;  at  100°  C.,  in 
the  vapor  apparatus  they  were  destroyed  in  a  few  minutes. 

Yersin  reports  that  when  fragments  of  the  spleen  or  liver  of 
animals  which  have  died  of  the  plague  are  fed  to  rats  and  mice  they 
usually  become  infected  and  die,  and  the  bacillus  is  found  in  their 
organs,  lymphatic  glands,  and  blood.  He  also  demonstrated  the  pres- 
ence of  the  bacilli  in  dead  rats  found  in  the  houses  or  streets  of 
Hong-Kong. 

167.  BACILLUS  PISCICIDUS   AGILIS  (Sieber). 

Discovered  by  Sieber  (1895)  in  infected  fish,  which  died  of  an  epidemic 
disease  in  the  laboratory  of  Professor  Nencki,  at  St.  Petersburg. 

Morphology. — Short  bacilli,  often  united  in  pairs. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  motile, 
liquefying  bacillus.  In  old  cultures  in  bouillon  spores  are  developed. 
Grows  at  temperatures  of  from  12°  to  37.5°  C.  Thermal  death  point,  00  to 
65°  C.  On  gelatin  and  agar  plates  forms  granular,  grayish,  or  yellowish 
colonies,  which  appear  to  be  made  up  of  three  concentric  rings — the  outer  one 
having  a  jagged  outline.  Gas  is  developed  during  the  growth  of  the  bacillus 
— carbon  dioxide  and  methyl  merecaptaii  in  small  amount.  Upon  potato  it 
f<»nns  yellowish-brown,  pearl -like  colonies.  Causes  coagulation  of  milk. 
Retains  its  vitality  and  virulence  for  months  in  well  or  river  water. 

Pathogenesis. — Pathogenic  for  fish,  frogs,  guinea-pigs,  rabbits,  mice, 
and  dogs  (not  for  birds).  Old  cultures  are  more  pathogenic  than  recent 
ones,  and  gelatin  cultures  are  the  most  active.  Frogs  are  killed  in  half  an 
hour  by  0.1  cubic  centimetre  of  a  bouillon  culture  six  days  old.  Filtered 
cultures  are  as  toxic  as  those  containing  the  living  bacillus  ;  they  give  with 


iro 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  523 

11  chloride  a  characteristic  color  reaction — an  intense  red  color.  Sieber 
has  obtained  from  his  cultures  an  extremely  toxic  alkaloid  in  the  form  of  a 
hydrochlorate.  Two  litres  of  filtered  culture  gave  0.1  gramme  of  this  salt. 
An  aqueous  solution  of  this  killed  a  frog  in  fifteen  minutes  in  the  dose  of 
0.0035  gramme. 

168.    BACILLUS   OF  MERESHKOWSKY. 

Obtained  by  Mereshkowsky  (1894)  from  infected  animals  (Spermophilus 
musicus)  which  died  from  an  epidemic  malady  developed  in  his  laboratory. 

Morphology. — Closely  resembles  Loffler's  Bacillus  typhi  murium. 

Biological  Characters.— An  aerobic,  motile,  non-liquefying  bacillus. 
Spore  formation  not  observed.  Grows  in  the  usual  culture  media  at  the 
room  temperature  — best  at  37.5°  C.  In  bouillon,  at  the  end  of  twenty-four 
hours,  the  medium  is  clouded  and  a  white  pellicle  is  seen  upon  the  surface, 
which  breaks  up  into  small  flocculi  and  falls  to  the  bottom  when  the  tube 
is  slightly  shaken.  On  gelatin  plates  minute,  slightly  granular,  pale-brown 
colonies  may  be  seen,  under  a  low  power  at  the  end  of  twenty-four  hours  ; 
on  the  sscond  day  these  are  visible  as  white  spheres,  which  under  the  micro- 
scope have  a  pale-brown  color  and  a  more  or  less  transparent,  peripheral 
zone.  In  media  containing  glucose  110  gas  is  developed.  The  growth  upon 
agar  and  potato  presents  nothing  characteristic. 

Pathogenesis. — Pathogenic  for  Zieselmausen  (Spermophilus  musicus), 
for  Spermophilus  guttatus,  for  squirrels  (Sciurus  vulgaris)  for  house  mice, 
for  field  mice  (Arvicola  arvalis).  Not  pathogenic  for  man  or  for  the  domes- 
tic animals  tested,  horse,  swine,  sheep,  fowls.  Mereshkowsky  proposes  to 
use  cultures  of  this  bacillus  for  the  extermination  of  field  mice,  which  die  in 
from  one  to  ten  days  after  being  fed  upon  biscuit  wet  with  a  bouillon  cul- 
ture. 

169.    BACILLUS   OF  EMMERICH   AND   WEIBEL. 

Obtained  by  Emmerich  and  Weibel  (1894)  from  infected  trout  in  ponds 
belonging  to  an  establishment  for  raising  these  fish.  The  disease  appeared 
as  a  superficial  ' '  f urunculosis  with  secondary  development  of  abscesses  con- 
taining bloody  pus."  Death  occurred  in  from  twelve  to  twenty  days.  The 
pustules  and  secondary  abscesses  and  blood  from  the  heart  and  various  or- 
gans contained  bacilli,  which  proved  to  be  the  cause  of  the  infectious  malady. 

Morphology. — Bacilli  about  as  long  as  the  typhoid  bacillus,  but  not  so 
thick,  very  frequently  united  in  pairs  ;  occasionally  grows  out  into  filaments. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  lique- 
fying, non-motile  bacillus.  Does  not  form  spores.  Thermal  death  point, 60° 
C.  Stains  with  the  usual  aniline  colors  but  not  by  Gram's  method.  Grows 
best  at  10°  to  15°  C.  The  growth  in  gelatin  is  quite  characteristic.  At  the 
end  of  two  or  three  days,  in  gelatin  plates,  at  the  room  temperature,  small 
white  colonies  are  developed  ;  in  four  or  five  days  small  gas  bubbles  or  ex- 
cavations are  seen,  at  the  bottom  of  which  lie  the  scale-like  or  rosetta-formed 
colonies.  The  margin  of  the  colonies  is  irregular  and  later  jagged.  At 
first  the  colonies  are  grayish- white  or  yellowish,  later  brownish.  The 
superficial  colonies  have  a  peculiar  lustre.  In  gelatin  stick  cultures,  colo- 
nies develop  along  the  line  of  puncture,  which  at  first  resemble  the  growth 
of  Streptococcus  pyogenes,  and  no  development  is  seen  on  the  surface.  At 
the  end  of  five  to  seven  days  in  place  of  the  line  of  colonies  is  seen  a  channel 
filled  with  air,  or  gas  developed  by  the  separate  colonies,  the  bubbles  from 
which  coalesce.  The  funnel  formed  in  this  way  is  somewhat  larger  above, 
and  at  the  bottom  contains  a  whitish  sediment  consisting  of  bacteria  con- 
tained in  a  few  drops  of  liquefied  gelatin.  Along  the  sides  of  the  funnel 
bubble-like  cavities  may  frequently  be  seen,  at  the  bottom  of  which  the  bac- 
teria have  accumulated.  In  bouillon  a  slight  cloudiness  is  seen  near  the 
surf  ace,  on  the  walls  of  the  test  tube;  when  .slightly  shaken  this  falls  to  the 


524  PATHOGENIC   AEROBIC   BACILLI 

bottom,  leaving-  the  bouillon  entirely  clear.  In  agar-agar  tubes,  a  veil- 
like  stripe  develops  along  the  line  of  puncture,  and  a  grayish-yellow,  moist 
layer,  with  irregular  outlines  upon  the  surface.  After  some  weeks  this 
acquires  a  brown  color.  No  growth  occurs  upon  potato.  No  development 
occurs  in  the  incubating  oven  at  37°  C. 

Pathogenesis. — Trout  became  infected  and  died  through  direct  infection, 
subcutaneous  or  intramuscular  inoculations,  or  through  the  addition  of  cul- 
tures to  the*  water  in  which  they  were  kept,  or  by  placing  infected  fish  in  the 
same  tank  with  healthy  ones. 

170.    GAS-FORMING   AEROBIC    BACILLUS   OF   LASER. 

Obtained  by  Laser  (1892)  from  a  piece  of  liver  and  lung  from  a  calf  which 
died  of  an  infectious  disease. 

Morphology. — "  Short  bacilli." 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile,  gas-producing  bacillus.  Stains  by  Gram's  method. 
Spore  formation  not  observed.  In  gelatin  stick  cultures  development  oc- 
curs on  the  surface  in  the  form  of  a  button-like  mass,  and  along  the  line  of 
puncture  colonies  are  formed  which  may  be  separate  below.  The  colonies 
in  gelatin  and  agar  plates  are  not  characteristic.  Upon  agar,  at  37°  C.  a 
slimy,  moist,  shining  layer  is  developed  which  covers  the  entire  surface.  In 
stick  cultures  in  gelatin  or  agar  containing  glucose  an  abundant  develop- 
ment occurs,  attended  with  an  evolution  of  gas.  In  bouillon,  in  the  incubat- 
ing oven,  a  uniform  cloudiness  of  the  culture  medium  is  seen  at  the  end  of 
twenty-four  hours,  and  the  bacilli  gradually  sink  to  the  bottom  of  the  tube. 
Upon  potato  in  the  incubating  oven,  a  shining,  white  layer  is  developed 
over  the  entire  surface ;  on  potato  kept  at  the  room  temperature  a  thick, 
grayish-yellow  layer  in  the  middle,  which  gradually  becomes  more  decidedly 
yellow,  while  the  potato  around  this  growth  has  at  first  a  violet  shimmer, 
and  later  an  intense  violet  color. 

Pathogenesis. — The  limited  number  of  experiments  made  on  mice,  rab- 
bits, and  guinea-pigs  resulted  in  the  death  of  some  of  the  animals,  while 
others  recovered.  (This  appears  to  be  a  bacillus  of  the  colon  group,  which 
differs  but  little  from  Bacillus  coli  communis.  G.  M.  S.) 

171.    BACILLUS  OF  BECK. 

Synonym. — Der  Bacillus  der  Brustseuche  beim  Kaninchen. 

Obtained  by  Beck  (1892)  from  rabbits  which  died  of  an  infectious  malady 
in  the  Institut  fiir  Infectionskrankheiten,  in  Berlin. 

Morphology. — Very  small  and  slender  bacilli,  about  twice  as  long  and 
twice  as  thick  as  the  influenza  bacillus  ;  somewhat  pointed  at  the  extremities ; 
show  a  tendency  to  grow  out  into  filaments. 

Biological  Characters. — An  aerobic  (strict)  non-liquefying,  non-motile 
bacillus.  Spore  formation  not  observed.  Grows  at  the  room- temperature 
and  more  vigorously  at  38°  C.  Does  not  stain  by  Gram's  method.  Thermal 
death  point,  50°  C.  (five  minutes).  Resists  desiccation,  at  the  room  tempera- 
ture, for  seventeen  days,  at  37°  C.  for  three  days. 

On  gelatin  plates,  at  the  end  of  forty-eight  hours,  small,  finely  granular, 
glass-like  colonies  are  developed  ;  older  colonies  have  a  pale-brown  appear 
ance.  In  gelatin  stick  cultures  a  granular  growth  of  a  white  color  is  seen 
along  the  line  of  puncture.  Upon  agar,  at  37°  C.,  an  abundant  development 
occurs  in  twenty -four  hours.  The  line  of  puncture  seen  from  above  is  gray- 
ish-white,  by  transmitted  light  bluish  and  porcelain-like  with  a  brownish 
tint.  On  agar  plates  the  colonies  have  a  yellowish-gray  appearance ;  the 
margin  of  the  finely  granular  colonies  is  sharply  defined.  In  agar  cultures 
several  days  old  the  colonies  are  sticky  and  may  be  picked  up  as  a  compact 
mass,  or  drawn  out  into  threads.  In  bouillon,  at  37°  C.,  there  is  a  slight 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS. 


525 


cloudiness  at  the  end  of  twenty-four  hours  ;  later  the  bouillon  is  clear  and  a 
white  sediment  is  seen  at  the  bottom  of  the  tube.  In  bouillon  cultures 
especially,  the  bacillus  grows  out  into  long  filaments. 

Pathogenesis. — From  0.25  to  1  cubic  centimetre  of  a  bouillon  culture 
injected  into  the  pleural  cavity  of  a  rabbit  caused  a  development  of  all  of 
the  symptoms  of  influenza  (Brustseuche)— viz. ,  elevation  of  temperature  at 
the  end  of  five  or  six  hours,  cough,  nasal  discharge,  dyspnoea,  and  death — 
usually  in  from  three  to  five  days.  The  autopsy  showed  a  distinct  pleuro- 

fneumonia  and  a  general  blood  infection  by  the  bacillus  in  question, 
njections  into  the  circulation  also  give  rise  to  the  symptoms  of  influenza, 
including  pneumonia,  and  to  death  at  the  end  of  from  ten  to  fourteen  days. 
Subcutaneous  injections  resulted  in  the  development  of  an  abscess  and  of  ex- 
tensive necrosis  of  the  tissues,  but  did  not  cause  a  general  blood  infection. 
Guinea-pigs  were  somewhat  less  susceptible  than  rabbits,  but  injections  into 
the  pleural  cavity  produced  similar  symptoms  and  death  at  a  later  date. 
White  mice  and  house  mice,  as  a  result  of  intra peritoneal  injections,  died 
within  two  or  three  days  from  general  blood  infection. 


172.    BACILLUS   BOVIS   MORBIFICANS 

Obtained  by  Basenau  (1893)  from  the  flesh  of  a  cow,  which  is  supposed  to 
have  died  from  puerperal  fever  and  was  condemned  by  the  inspector  at  the 
slaughter-house  in  Amsterdam. 

Morphology. — Short  bacilli,  with  rounded  ends,  two  to  two  and  one-half 
times  as  long  as  broad,  usually  united  in  pairs.  0.3  to  0.4  ^  broad. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  actively  motile  bacillus.  Does  not  stain  by  Gram's  method. 
Does  not  form  spores — is  killed  in  one  minute  by  exposure  to  a  temperature 
of  70°  C.  Does  not  coagulate  milk.  In  media  containing  glucose  causes  a 
moderate  development  of  gas.  Grows  at  a  temperature  of  9°  C.,  best  in 
incubating  oven  at  37°  C.  In  bouillon,  at  37°  C.,  a  uniform  clouding  of  the 
medium  occurs  in  twenty-four  hours  ;  later,  a  thin,  smooth  pellicle  forms 
011  the  surface,  this  is  readily  broken  up  by  gentle  agitation  and  falls  to  the 
bottom,  where  a  grayish- white  mass  accumulates. 

In  gelatin  stick  cultures  a  slender,  yellowish-white  growth  is  seen  along 
the  line  of  puncture,  and  a  white,  thick  layer,  with  more  or  less  irregular 
outlines,  is  slowly  developed  on  the  surface.  In  streak  cultures  the  growth  is 
like  that  of  the  ' '  colon  bacillus. "  Upon  agar,  at  37°  C.,  at  the  end  of  twenty- 
four  hours,  an  abundant  grayish-white  layer  is  developed.  Upon  potato  it 
grows  more  slowly  and  forms  a  soft,  yellow  layer,  which  never  acquires  a 
brown  color. 

Pathogenesis. — Causes  a  fatal  infection  in  mice,  white  rats,  guinea-pigs, 
rabbits,  and  calves.  Mice  and  guinea-pigs  succumb  to  subcutaneous  injec- 
tions, rabbits  to  intra-peritoiieal  infection,  and  calves  to  intraperitoneal  injec- 
tions, or  from  the  iiigestion  of  milk  containing  the  bacilli.  Young  guinea- 
pigs  may  be  infected  through  the  mother's  milk.  (This  bacillus  belongs  to 
the  "colon  group"  and  is  probably  a  pathogenic  variety  of  Bacillus  coli 
communis. — G.  M.  S.) 


173.  BACILLUS  PISCICIDUS  (Fischel  and  Enoch). 

Obtained  by  Fischel  and  Enoch  (1892)  from  an  infected  carp. 
Morphology. — Bacilli  solitary  or  in  chains  of  four  to  five  elements, 
long-  and 


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to  3/*  long  and  0.25//  thick.     Stains  by  the  usual  aniline  colors  and  by 
Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile,  liquefying  bacillus.  Forms  spores.  In  gelatin  plates  forms  round 
colonies  of  a  pale  yellowish-brown  color,  having  a  slightly  toothed  border 
and  a  granular  surface.  At  the  end  of  twenty-four  hours  a  narrow  zone  of 


526  PATHOGENIC   AEROBIC   BACILLI 

liquefaction  can  be  discerned  around  the  colonies,  and  at  the  end  of  about 
ten  days  the  gelatin  is  entirely  liquefied.  In  gelatin  stick  cultures  a  scanty 
growth  is  seen  along  the  line  of  inoculation  at  the  end  of  twelve  hours  ;  the 
growth  upon  the  surface  is  rapid,  and  liquefaction  commences  at  the  end  of 
twenty-four  hours.  Upon  agar,  at  37°  C.  at  the  end  of  eighteen  hours,  a  thin 
granular  layer  is  seen,  which  consists  of  small,  pale-gray  colonies.  In  agar 
stick  cultures  a  scanty  growth  occurs  along  the  line  of  puncture,  which  does 
not  increase  after  thirty-six  hours.  Upon  the  surface  the  growthis  abundant, 
forming,  at  the  end  of  five  days  a  tolerably  thick  grayish-white  layer.  No 

growth  occurs  upon  potato  at  the  room  temperature,  but  at  37°  C.  a  tolera- 
ly  thick,  sticky  layer  of  a  grayish-white  color  is  developed  in  three  or  four 
days.  In  bouillon,  at  37°  C.,  the  medium  is  clouded  at  the  end  of  twelve 
hours,  and  a  thin  pellicle  is  seen  upon  the  surface  at  the  end  of  thirty-six 
hours ;  this  falls  to  the  bottom  when  the  tube  is  slightly  agitated.  At  the 
end  of  four  days  development  has  ceased,  and  the  bouillon  is  again  transpar- 
ent, while  a  flocculent  deposit  is  seen  at  the  bottom  of  the  tube.  The  bouillon 
gives  off  a  penetrating  odor,  like  that  of  burnt  milk.  The  same  odor  is  given 
off  from  cultures  in  milk,  which  is  peptonized  by  the  action  of  the  bacillus. 
At  the  end  of  twenty  days,  at  37°  C.,  the  entire  contents  of  the  tube  have  be- 
come transparent. 

Pathogenesis.  —Produces  a  fatal  infectious  disease  in  fish  ("gold  carp'1) 
when  inoculated  beneath  the  skin  ;  also  pathogenic  for  mice  and  for  guinea- 
pigs. 

174.    BACILLUS  PYOGENES  FILIFORMIS  (Flexner). 

Obtained  by  Flexner  (1895)  from  the  interior  of  the  uterus  and  from  an 
exudate  in  the  pericardial  and  pleural  cavities,  of  a  rabbit  which  died  on  the 
fifth  day  after  parturition! 

Morphology.—  Pleomorphous  cocci-like  forms,  short  or  long  bacilli,  and 
long  threads  are  seen  in  cover  slips  prepared  from  the  exudate.  "Very  few 
of  the  bacilli  stain  regularly  ;  for  the  most  part  brightly  stained  spots  appear 
between  stained  areas.  An  outer  membrane  always  stains,  enclosing  the 
stained  dots  in  a  colorless  ground.  The  threads,  as  a  rule,  present  delicate, 
sinuous,  and  wavy  outlines ;  the  short  forms  are  straight  with  rounded  ends." 

Biological  Characters. — All  attempts  to  cultivate  this  bacillus  in  the 
usual  media,  either  in  the  presence  of  oxygen  or  in  an  atmosphere  of  hydro- 
gen, proved  unsuccessful.  But  successive  cultures  were  made  by  inoculations 
in  the  pleural  cavity  of  rabbits — a  bit  of  pleural  exudate  suspended  in  bouil- 
lon was  used  for  this  purpose.  The  bacillus  was  also  propagated  upon  the 
lungs,  heart,  uterus,  and  kidney  of  healthy  rabbits.  The  organs  were  re- 
moved with  great  care  to  prevent  contamination  and  placed  in  sterilized  test 
tubes.  Transplantations  from  these  cultures  were  only  successful  for  one  or 
two  generations.  Better  results  were  obtained  by  cultivating  the  bacillus 
upon  the  one-third  to  one-half  grown  foetuses  of  rabbits. 

Pathogenesis. — "  Considerable  variations  were  observed  according  as  the 
inoculations  were  made  into  the  pleural  cavity,  the  peritoneal  cavity,  the  sub- 
cutaneous tissue,  beneath  the  dura  mater,  or  directly  into  the  circulation. 
The  inoculations  gave  positive  results  in  all  cases  except  a  few,  in  which  they 
were  made  subcutaneously.  The  death  of  the  animal  occurred  soonest  when 
inoculation  was  made  beneath  the  dura  mater.  A  small  portion  of  the  skull 
was  trephined,  care  being  taken  to  exclude  rxt raucous  microorganisms,  and 
a  drop  of  the  pleural  fluid  or  a  speck  of  the  librinous  oxiidato  \vas  introduced 
beneath  (lie  membranes,  care,  being1  taken  not  to  injure  the  brain.  These 
animals,  which  quickly  recovered  from  the  effects  of  the  operation,  died  on 
an  average  about  twelve  hours  after  the  inoculation.  .  .  . 

"  The  pleural  inoculations  were  followed  by  death,  as  before  stated,  in  ev- 
ery instance,  the  death  of  the  animal  occurring  upon  the  third  or  fourth  day. 
The  appearances  presented  at  the  autopsy  were  for  the  most  part  an  exact 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  527 

reproduction  of  those  observed  in  the  animal  which  had  succumbed  to  the 
natural  disease.  Upon  the  side  of  inoculation  a  thick,  grayish-yellow,  shaggy 
membrane  covered  the  pleural  surfaces,  being  at  times  four  or  five  millime- 
tres in  thickness.  The  pleural  cavity  contained  several  cubic  centimetres  of 
a  clear  haemoglobin-colored  fluid,  the  lung  for  the  most  part  being  com- 
pressed. At  times  smaller  or  larger  areas  of  lobular  pneumonia  would  be 
present ;  and,  as  a  rule,  the  inflammation  was  not  limited  to  the  serous  mem- 
brane of  the  side  of  inoculation,  but  extended  into  the  opposite  pleural  cavity 
and  into  the  pericardial  sac.  However,  in  these  situations  the  process  was, 
as  a  rule,  less  intense,  the  solid  exudate  being  less  considerable,  and  in  the 
case  of  the  opposite  pleural  cavity  sometimes  entirely  wanting.  The  super- 
ficial vessels,  however,  were  injected  and  the  serous  surface  of  the  affected 
membrane  covered  with  a  slimy,  clear  fluid.  In  addition  to  this  the  oppo- 
site pleural  cavity  always  contained  a  similar  pink  serum  to  that  described 
upon  the  side  of  inoculation. 

"The study  of  the  exudate  upon  the  side  of  inoculation  as  well  as  the 
fluid  contained  in  the  opposite  pleural  cavity  and  in  the  pericardium  showed 
the  same  organisms  as  had  been  introduced." 

175.    BACILLUS   OF  UNNA  AND   HODARA. 

Obtained  by  Hodara  (1894)  from  the  contents  of  acne  pustules — **  in  enor- 
mous masses  in  the  comedones  of  true  acne." 

Morphology.—  Small  bacilli,  from  0.3  to  0.7  t*  long  and  0.3  ft  thick. 
When  stained,  by  Unna's  method,  with  methylene-blue-glycerin  ether,  or  with 
methylene-blue-tannin  solution,  they  are  seen  to  be  surrounded  by  a  homo- 
geneous, jelly-like  mass  which  is  stained  pale  violet  by  the  first  method  and 
green  by  the  second.  The  bacilli  are  sometimes  united  in  chains  of  two  or 
three  elements,  and  single  rods  may  present  in  the  middle  an  unstained  zone 
with  deeply  stained  extremities. 
Biological  Characters.—  Not  determined. 

176.  PROTEUS  FLUORESCENS  (Jaeger). 

Obtained  by  Jaeger  (1892)  from  the  liver,  spleen,  and  kidneys  of  fatal 
cases  of  infectious  icterus  ("Weil's  disease"). 

Morphology.— A.  pleomorphous  bacillus  of  theproteus  group  ;  in  the  same 
culture  cocci-like  elements,  short  rods  either  straight  or  curved,  and  long 
filaments  are  seen. 

Biological  Characters. — An  aerobic  and  facultat ive  anaerobic,  motile, 
liquefying,  chromogenic  bacillus.  Is  not  to  be  distinguished  from  Proteus 
vulgaris  except  by  the  fact  that  it  produces  an  intense  fluorescent-green  pig- 
ment. Jaeger  says  that  cultures  which  originally  failed  to  liquefy  gelatin  and 
produced  the  fluorescent-green  pigment,  at  the  end  of  two  and  a  half  years 
had  lost  the  property  of  producing  pigment  and  had  acquired  the  property  of 
liquefying  gelatin,  and  could  not  be  distinguished  from  Proteus  vulgaris. 
But  when  these  cultures  were  kept  at  a  lower  temperature  they  gradually 
regained  their  former  characters. 

Pathogenesis.—  Pathogenic  for  mice  and  for  pigeons;  but  the  virulence 
of  cultures  proved  to  be  very  variable. 

177.    MICROCOCCUS  INSECTORUM   (Burrill). 

Obtained  by  Burrill  (1883)  from  the  alimentary  canal  of  infected  "chinch 
bugs"  (Blissus  leucopterus).  , 

Morphology.—  Oval  or  spherical  (micrococci  ?)  bacteria,  usually  in  pairs, 
but  sometimes  in  chains  of  four  to  eight  elements.  ' '  Undivided  segments  vary 
from  0.8  to  1.6  /«  in  length,  with  a  uniform  width  of  0.65  p. "  (Forbes). 


528  PATHOGENIC  AEROBIC   BACILLI 

Biological  Characters.— Forbes  (1891)  says:  "I  have  lately  succeeded, 
in  conjunction  with  Professor  Burrill,  in  making  pure  cultures  in  consid- 
erable numbers  in  both  animal  and  vegetable  media."  .  .  .  "We  have  ob- 
tained successful  cultures  in  all  the  neutral  and  alkaline  fluids  and  in  none 
of  the*acid  ones."  Non-motile  and  does  not  form  spores. 

178.    BACILLUS  MONACH^E  (v.    Tubeuf). 

Obtained  by  v.  Tubeuf  (1892)  from  infected  caterpillars  of  Liparis 
monacha. 

Morphological  and  Biological  Characters. — Short,  motile,  aerobic,  non- 
liquefying  bacilli,  which  grow  in  the  usual  culture  media  at  the  room  tem- 
perature. 

179.    MICROCOCCUS   OF  BRUCE. 

Obtained  by  Bruce  (1892)  from  the  spleen,  post-mortem — of  cases  of  so- 
called  "Malta  fever." 

Morphology. — Micrococci,  about  .33  p  in  diameter,  solitary  or  in  pairs — 
never  in  chains. 

Biological  Characters. — An  aerobic,  non-liquefying,  micrococcus.  Does 
not  stain  by  Gram's  method.  Grows  best  in  nutrient  agar.  In  stab  cultures 
no  growth  is  seen  for  several  days.  "At  length  the  growth  appears  as 
pearly- white  spots  scattered  around  the  point  of  puncture  and  minute,  round, 
white  colonies  are  also  seen  along  the  course  of  the  needle  track "  ;  these 
increase  in  size  and  after  some  weeks  a  rosette-shaped  growth  is  seen  upon 
the  surface,  and  the  growth  along  the  line  of  puncture  has  a  yellowish-brown 
color.  At  the  end  of  nine  or  ten  days,  at  37°  C.,  some  of  the  colonies  on  the 
surface  of  nutrient  agar  are  as  large  as  No.  4  shot;  by  transmitted  light 
they  have  a  yellowish  color  at  the  centre,  and  the  periphery  is  bluish-white ; 
by  reflected  light  they  have  a  milky-white  color.  At  25°  C.  colonies  first 
become  visible  at  the  end  of  about  seven  days,  at  37°  C.  in  three  to  four 
days.  Does  not  grow  upon  potato.  Very  scanty  growth  upon  nutrient 
gelatin  at  22°  C.  at  the  end  of  a  month. 

Pathogenesis. — Pathogenic  for  monkeys,  which  suffer  from  fever  as  a 
result  of  subcutaneous  inoculations  and  usually  (three  out  of  four  experi- 
mented upon)  die  in  from  thirteen  to  twenty-one  days.  The  spleen  is  found 
to  be  enlarged  and  contains  the  micrococcus.  Not  pathogenic  for  mice, 
guinea-pigs,  or  rabbits. 

180.    BACILLI  OF  GUILLEBEAU  (a,  6,  and  C). 

Obtained  by  Guillebeau  from  the  milk  of  cows  suffering  from  mastitis, 
and  found  by  Freudenreich  to  produce  an  abnormal  fermentation  of  cheese, 
characterized  by  the  presence  of  large  cavities  ("boursouflement")  and  by  a 
very  bad  taste. 

BACILLUS  a. 

Morphology. — Varies  considerably  in  size,  and  may  resemble  a  micrococ- 
cus in  form  ;  usually  1  //  broad  and  1  to  2  ft-  long. 

Stains  with  the  usual  aniline  colors,  but  rather  feebly  ;  does  not  stain  by 
Gram's  method. 

Biological  Characters.—  A.naerobics,ndfact<lf<ifir<>  anaerobic,  slightly 
motile,  non-liquefyingl>a<ci\]us.  Spore  formation  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  the 
deep  colonies  are  spherical,  granular,  and  yellowish  in  color  ;  upon  the  sur- 
face they  are  round  and  granular  at  first,  later  they  become  opaque  and  re- 
semble a  drop  of  wax.  In  gelatin  stick  cultures  development  occurs  all 
along  the  line  of  puncture,  and  upon  the  surface  a  whitish  layer  is  formed. 


NOT   DESCRIBED   IX   PREVIOUS   SECTIONS.  529 

Upon  agar  a  grayish-white  layer  is  developed.  Upon  potato  a  thick,  yel- 
lowish layer  is  formed  ;  this  is  viscid  and  contains  numerous  gas  bubbles. 
In  milk  coagulation  is  produced  at  the  end  of  twenty-four  hours,  and  an 
abundance  of  gas  is  given  off.  In  bouillon  containing  milk  sugar  it  multi- 
plies abundantly  and  a  large  quantity  of  gas  is  liberated.  Grows  best  at  a 
temperature  of  30'  to  35°  C.  Thermal  death-point  60°  C.—  fifteen  minutes' 
exposure. 

BACILLUS  b. 

Morphology.  —  Resembles  bacillus  a  ;  bacilli  from  1  to  2  //  long  and  about 
1  n  thick. 

Biological  Characters.  —  Anaerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Is  differentiated  from  a  by  the  fact  that  it  causes  lique- 
faction of  nutrient  gelatin  after  an  interval  of  several  weeks,  and  by  the  fact 
that  the  young  colonies  upon  gelatin  plates  are  quite  viscid.  Spore  forma- 
tion not  observed.  Thermal  death-point  80°  C.  —  five  minutes'  exposure. 
An  abundance  of  gas  is  given  off  from  cultures  containing  milk  sugar. 

BACILLUS  C. 

Morphology.  —  Short  bacilli  ;  often  oval  or  even  spherical  in  form  ;  about 
1  n  long. 

Biological  Characters.  —  An  aerobic,  non-liquefying  bacillus.  Spore 
formation  not  observed.  Upon  gelatin  plates  colonies  are  developed  which 
resemble  those  of  bacillus  a,  but  are  more  closely  granular  ;  the  colonies 
are  very  adherent  and  difficult  to  remove  from  the  culture  medium.  Upon 
agar  a  viscous,  white  layer  is  developed.  Upon  potato  the  growth  is  of  a 
yellowish-  white  color  and  similar  so  that  of  a  and  b,  with  gas  bubbles  ;  it  is 
very  adherent.  In  liquid  media  the  growth  of  this  bacillus  causes  the  cul- 
ture liquid  to  become  extremely  viscous  and  almost  gelatinous  in  consistence. 
In  milk  coagulation  occurs  at  the  end  of  sixty  hours  at  37°  C.,  and  the  milk 
then  loses  its  viscosity. 

181.    BACILLUS  AEROGENES  MENINGITIDIS    (Centanni). 

Found  by  Centanni  (1893)  in  two  fatal  cases  of  meningitis. 

Morphology.—  Bacilli  from  2  to2.5/^  long  and  £//  thick,  with  rounded 
extremities  ;  solitary,  in  pairs  or  in  short  chains. 

Biological  Characters.  —  An  aerobic  and  facultative  anaerobic,  motile, 
liquefying  bacillus.  Spore  formation  not  demonstrated.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  In  gelatin  stick  cultures  len- 
ticular gas  bubbles  are  developed  ;  gas  bubbles  are  also  developed  in  profu- 
sion in  the  abundant  growth  upon  the  surface  of  cooked  potato. 

Pathogenic  for  rabbits. 


182.  PNEUMO-BACILLUS  SEPTicus  (Galtier). 

Found  by  Galtier  in  the  pulmonary  exudate,  etc.,  in  calves  suffering 
from  the  infectious  pleuro-pneumonia,  or  pneumo-enteritis,  to  which  it  gives 
rise. 

Morphology.  —  Spherical,  oval,  or  rod-shaped  bacteria,  usually  in  pairs, 
sometimes  in  short  chains.  The  rods  are  sometimes  three  or  four  times  as 
long  as  broad,  with  round  ends.  Stains  with  the  usual  aniline  colors,  but 
not  by  Gram's  method. 

Biological  Characters.—  A  motile,  aerobic,  and  facultative  anaerobic, 

non-liquefying  bacillus.     (It  does  not  liquefy  gelatin  under  ordinary  con- 

ditions,  but  when  recently  prepared  and  containing  less  than  the  usual 

amount  of  gelatin,  liquefaction  may  occur.)     Grows  rapidly  in  the  usual 

37 


530  PATHOGENIC   AEROBIC  BACILLI 

culture  media  at  the  room  temperature — still  more  rapidly  at  37°  C.     The 
cultures  give  off  a  peculiar  odor.     Forms  spores. 

Pathogenesis. — According-  to  Galtier  injections  into  the  lungs  or  into  the 
trachea,  in  calves,  pigs,  rabbits,  or  guinea-pigs,  cause  a  development  of  the 
disease,  "with  a  predominance  of  the  pleuro-pulmonary  lesions." 

183.    BACILLUS  PSEUDO-TUBERCULOSIS  OF  PREISZ. 

Obtained  by  Preisz  (1894)  from  an  infected  sheep. 

Morphology. — Resembles  the  bacillus  of  diphtheria,  but  is  smaller. 

Biological  Characters. — An  aerobic,  and  facultative  anaerobic,  non- 
motile  bacillus.  Does  not  |pow  in  nutrient  gelatin  at  the  room  temperature. 
In  bouillon  a  scaly  pellicle  is  formed  upon  the  surface  which  breaks  up  upon 
slight  agitation ;  the  bouillon  is  but  slightly  clouded.  Upon  blood  serum 
the  colonies,  at  37°  C.,  have  a  golden  or  orange-yellow  color  ;  this  varies  con- 
siderably in  different  cultures.  Does  not  form  pigment  in  agar  cultures, 
does  not  grow  upon  potato.  Stains  by  Gram's  method. 

Pathogenesis. — Pure  cultures  inoculated  into  rabbits  or  guinea-pigs  give 
rise  to  a  pseudo-tuberculosis  of  the  lymphatic  glands,  the  spleen,  the  liver, 
the  mesentery,  etc.  This  ends  fatally  in  from  ten  to  thirty-five  days. 

184.   BACILLUS  PSEUDO-TUBERCULOSIS  MURIUM. 

Obtained  by  Kutscher  (1894)  from  a  mouse  which  died  in  the  laboratory. 

Morphology. — Slender  bacilli,  which  frequently  have  pointed  extremities, 
about  the  length  of  the  diphtheria  bacillus,  and  like  this  bacillus  quite  vari- 
able in  form. 

Biological  Characters.  — An  aerobic  and  facultative  anaerobic,  non- 
motile,  non-liquefying  bacillus.  Spore  formation  not  demonstrated.  Stains 
by  Gram's  method.  Upon  agar  plates  small  yellowish  colonies  are  devel- 
oped at  the  end  of  twenty-four  hours,  at  37°  C.  ;  these  have  a  finely  granular 
centre  and  a  dentate  margin  ;  between  the  sharply  dentate  processes  are  seen 
short,  relatively  thick  projections.  The  superficial  colonies  are  in  the  form 
of  delicate,  transparent,  white  layers  ;  these  resemble  colonies  of  Streptococcus 
pyogenes.  They  reach  the  limit  of  their  development  in  four  or  five  days. 
Upon  gelatin  plates  similar  colonies  are  developed,  which  become  visible  at 
the  end  of  forty-eight  hours  and  continue  to  increase  in  size  for  twelve  to  fif- 
teen days.  In  bouillon  a  slight  clouding  of  the  culture  medium  occurs  at 
the  end  of  twenty -four  to  fortv-eight  hours,  and  later  a  finely  granular  de- 
posit is  seen  at  the  bottom  of  the  tube ;  upon  the  surface  a  thin  pellicle  is 
formed,  made  up  of  coffin-shaped  crystals.  In  milk  the  growth  is  abundant, 
but  does  not  cause  any  perceptible  change  in  the  culture  medium.  Upon 
potato  no  development  occurs. 

Pathogenesis.— Pathogenic  for  mice,  in  which  pseudo-tubercles  of  the 
lungs  are  developed  as  a  result  of  the  subcutaneous  injection  of  a  small 
amount  of  a  pure  culture.  Injections  into  the  peritoneal  cavity  are  fatal 
in  from  three  to  five  days.  Not  pathogenic  for  rabbits  or  guinea-pigs. 


XIV. 
PATHOGENIC  ANAEROBIC  BACILLI. 

STRICTLY  anaerobic  bacilli  are  not  able  to  multiply  in  the  blood 
of  living  animals  ;  but  some  of  them  may  multiply  in  the  subcuta- 
neous connective  tissue  or  in  the  muscles,  when  introduced  by  in- 
oculation, and  are  pathogenic  because  of  the  local  inflammatory  or 
necrotic  processes  to  which  they  give  rise,  or  because  they  produce 
soluble  toxic  substances  which  are  absorbed  and  cause  death  by 
their  special  action  upon  the  nervous  system  or  by  general  toxaemia. 

185.    BACILLUS  TETANI. 

Synonyms. — The  bacillus  of  tetanus  ;  Tetanusbacillus,  Ger. 

Nicolaier  (1884)  produced  tetanus  in  mice  and  rabbits  by  intro- 
ducing garden  earth  beneath  their  skin,  and  showed  that  the  disease 
might  be  transmitted  to  other  animals  by  inoculations  with  pus  or 
cultures  in  blood  serum  containing  the  tetanus  bacillus,  which,  how- 
ever, he  did  not  succeed  in  obtaining  in  pure  cultures.  Carle  and 
Rattone  (1884)  showed  that  tetanus  is  an  infectious  disease,  which 
may  be  transmitted  by  inoculation  from  man  to  lower  animals — a 
fact  which  has  since  been  verified  by  the  experiments  of  Rosenbach 
and  others.  Obtained  in  pure  cultures  by  Kitasato  (1889). 

The  writer  produced  tetanus  in  a  rabbit  in  1880  by  injecting  be- 
neath its  skin  a  little  mud  from  the  street  gutters  in  New  Orleans. 
The  tetanus  bacillus  appears  to  be  a  widely  distributed  microorgan- 
ism in  the  superficial  layers  of  the  soil  in  temperate  and  especially  in 
tropical  regions.  In  Nicolaier's  experiments  it  was  not  found  in  soil 
from  forests  or  in  the  deeper  layers  of  garden  earth. 

Morphology. — Slender,  straight  bacilli,  with  rounded  ends, 
which  may  grow  out  into  long  filaments.  Spores  are  developed  at 
one  extremity  of  the  bacilli,  which  are  spherical  in  form  and  consid- 
erably greater  in  diameter  than  the  rods  themselves,  giving  the 
spore-bearing  bacilli  the  shape  of  a  pin. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 
The  method  of  Ziehl  may  be  employed  for  double-staining  bacilli  and 
spores. 


532 


PATHOGENIC   ANAEROBIC   BACILLI. 


Biological  Characters. — An  anaerobic,  liquefying,  motile 
bacillus.  Forms  spores.  Grows  at  the  room  temperature,  in  the 
absence  of  oxygen,  in  the  usual  culture  media.  Grows  best  at  a 
temperature  of  36°  to  38°  0.;  in  nutrient  gelatin,  at  20°  to  25°  C., 
development  is  first  seen  at  the  end  of  three  or  four  days  ;  does  not 
grow  at  a  temperature  below  14°  C.  Spores  are  formed  in  cultures 
kept  in  the  incubating  oven  at  36°  C.,  at  the  end  of  thirty  hours  ; 
in  gelatin  cultures  at  20°  to  25°  C.,  at  the  end  of  a  week  (Kitasato). 
The  bacilli  exhibit  voluntary  movements  which  are  not  very  active  ; 
those  containing  spores  are  not  motile.  It  may  be  cultivated  in  an 
atmosphere  of  hydrogen,  but  does  not  grow  in  the  presence  of  oxy- 
gen— strictly  anaerobic — or  in  an  atmosphere  of  carbon  dioxide. 
The  addition  of  one  and  one-half  to  two  per  cent  of  grape  sugar  to 
nutrient  agar  or  gelatin  causes  the  development  to  be  more  rapid 


FIG.  161. 


FIG.  162. 


FIG.  161.— Tetanus  bacillus,  from  a  gelatin  culture,  x  1,000.  From  a  photomicrograph  by 
Pfeiffer. 

FIG.  162.— Tetanusbacillus,  from  an  agar  culture  ;  spore-bearing  rods,  x  1,000.  From  a  photo- 
micrograph by  Pfeiffer. 

and  abundant.     The  culture  medium  should  have  a  feebly  alkaline 
reaction. 

Colonies  in  gelatin  plates,  in  an  atmosphere  of  hydrogen,  re- 
semble somewhat  colonies  of  Bacillus  subtilis,  the  opaque  central 
portion  being  surrounded  by  a  circle  of  diverging  rays  ;  liquefaction 
is,  however,  much  slower,  and  the  resemblance  is  lost  after  a  short 
time.  Older  colonies  resemble  the  colonies  of  certain  microscopic 
fungi,  being  made  up  of  diverging  rays.  In  long  gelatin  stick  cul- 
tures development  occurs  along  the  line  of  puncture,  at  a  consid- 
erable distance  below  the  surface,  in  the  form  of  a  radiate  out- 
growth ;  the  gelatin  is  slowly  liquefied,  and  a  small  amount  of  gas  is 
at  the  same  time  formed.  In  peptonized  bouillon  having  a  slightly 
alkaline  reaction,  under  hydrogen  gas,  the  development  is  abundant 


PATHOGENIC   ANAEROBIC   BACILLI. 


533 


and  the  cultures  give  off  a  characteristic  odor — "  brenzlichen  Ge- 
ruch  "  (Kitasato). 

According  to  Kitasato,  blood  serum  is  not  a  very  favorable  me- 
dium for  the  growth  of  the  tetanus  bacillus,  and — contrary  to  the 
statement  of  Kitt,  Tizzoni,  and  others — 
it  does  not  cause  liquefaction  of  this 
medium. 

The  spores  of  the  tetanus  bacillus  re- 
tain their  vitality  for  months  in  a  desic- 
cated condition,  and  are  not  destroyed  in 
two  and  one-half  months  when  present 
in  putrefying  material  (Turco).  They 
withstand  a  temperature  of  80°  C.  main- 
tained for  an  hour,  but  are  killed  by 
five  minutes5  exposure  to  steam  at  100°  C. 
They  are  not  destroyed  in  ten  hours  by 
a  five-per-cent  solution  of  carbolic  acid, 
but  did  not  grow  after  fifteen  hours'  ex- 
posure in  the  same  solution.  A  five- 
per-cent  solution  of  carbolic  acid,  to 
which  0. 5  per  cent  of  hydrochloric  acid 
has  been  added,  destroys  them  in  two 
hours  ;  in  sublimate  solution  containing 
1  : 1,000  of  mercuric  chloride  they  are 
destroyed  at  the  end  of  three  hours,  or 
in  thirty  minutes  when  0.5  per  cent  of 
hydrochloric  acid  is  added  to  the  solu- 
tion. Kitasato  succeeded  in  obtaining 
pure  cultures  from  the  pus  formed  in 
the  vicinity  of  inoculation  wounds,  by 
destroying  the  associated  bacilli  after 
the  tetanus  bacilli  had  formed  spores. 

This  was  effected  by  heating  cultures  from  this  source  for  about  an 
hour  at  a  temperature  of  80°  C.  The  spores  of  the  tetanus  bacillus 
survived  this  exposure,  and  colonies  were  obtained  from  them  in  flat 
flasks  especially  devised  for  anaerobic  cultures  ;  from  these  colonies 
pure  cultures  in  nutrient  agar  or  gelatin — long  stick  cultures — or  in 
peptonized  bouillon  were  easily  obtained. 

Brieger  (1886)  first  succeeded  in  obtaining  from  impure  cultures 
of  the  tetanus  bacillus  a  crystallizable  toxic  substance,  called  by  him 
tetanin,  which  was  found  to  kill  small  animals  in  very  minute  doses 
and  with  the  characteristic  symptoms  of  tetanus.  More  recently 
Kitasato  and  Weyl  have  obtained  the  same  substance,  by  following 
Brieger's  method,  from  a  pure  culture  of  this  bacillus.  From  a 


Fio.  163.— Culture  of  Bacillus  tetani 
in  nutrient  gelatin.    (Kitasato.) 


534  PATHOGENIC   ANAEROBIC   BACILLI. 

bouillon  made  from  one  and  one-fourth  kilogrammes  of  lean  beef,  with 
the  addition  of  twenty-five  grammes  of  peptone,  they  obtained  1.7118 
grammes  of  hydrochlorate  of  tetanin.  This  proved  fatal  to  white 
mice  in  six  hours  in  the  dose  of  0.05  gramme,  and  a  dose  of  0.105 
gramme  caused  characteristic  tetanic  convulsions  and  death  within 
an  hour.  The  bacteriologists  last  named  also  obtained  from  their 
cultures  the  tetanotoxin  of  Brieger.  Two  mice  were  inoculated  sub- 
cutaneously  with  0.003  gramme  of  this  substance  ;  one  died  at  the 
end  of  five  hours  without  the  development  of  tetanic  symptoms  ; 
the  other  survived.  In  addition  to  these  substances,  indol,  phenol, 
and  butyric  acid  were  demonstrated  to  be  present  in  cultures  of  the 
tetanus  bacillus. 

According  to  Kitasato,  the  tetanus  bacillus  does  not  become  at- 
tenuated in  its  pathogenic  potency  by  cultivation  in  artificial  media, 
as  is  the  case  with  many  other  pathogenic  bacteria.  The  more 
recent  researches  of  Brieger  and  Frankel,  and  of  Kitasato,  show  that 
the  toxic  ptomaine  discovered  by  Brieger  in  1886  is  not  the  substance 
to  which  cultures  of  the  tetanus  bacillus  owe  their  great  and  pecu- 
liar pathogenic  power.  The  distinguished  German  chemist  and  his 
associate  have  succeeded  in  isolating  from  tetanus  cultures  a  toxal- 
bumin  which  is  far  more  deadly  than  tetanin. 

Pathogenesis. — The  experiments  of  Kitasato  (1889)  show  that 
pure  cultures  of  the  tetanus  bacillus  injected  into  mice,  rabbits,  or 
guinea-pigs  produce  typical  tetanic  symptoms  and  death.  As  the 
presence  of  this  bacillus  at  the  seat  of  injury,  in  cases  of  tetanus  in 
man,  has  now  been  demonstrated  by  numerous  observers,  there  is 
no  longer  any  question  that  tetanus  must  be  included  among  the 
traumatic  infectious  diseases,  and  that  the  bacillus  of  Nicolaier  and 
of  Kitasato  is  the  specific  infectious  agent.  Kitasato's  recently  pub- 
lished experiments  (1890)  show  that  cultures  of  the  tetanus  bacillus 
which  have  been  sterilized  by  filtration  through  porcelain  produce 
the  same  symptoms,  and  death,  in  the  animals  mentioned,  as  result 
from  inoculation  with  cultures  containing  the  bacillus.  It  is  evi- 
dent, therefore,  that  death  results  from  the  action  of  a  toxic  sub- 
stance produced  by  the  bacillus.  This  is  further  shown  by  the  fact 
that  the  bacillus  itself  cannot  be  obtained  in  cultures  from  the  blood 
or  organs  of  an  animal  which  has  succumbed  to  an  experimental  in- 
oculation with  an  unfiltered  culture;  but  the  blood  of  an  animal 
killed  by  such  an  inoculation  contains  the  tetanus  poison,  and  when 
injected  into  a  mouse  causes  its  death  with  tetanic  symptoms. 

When  a  platinum  needle  is  dipped  into  a  pure  culture  of  the 
tetanus  bacillus  and  a  mouse  is  inoculated  with  it  subcutaneously, 
the  animal  invariably  falls  sick  within  twenty-four  hours  and  dies  of 
typical  tetanus  in  two  or  three  days.  Rats,  guinea-pigs,  and  rabbits 


PATHOGENIC   ANAEROBIC   BACILLI.  535 

are  killed  in  the  same  way  by  somewhat  larger  quantities — 0.3  to  0.5 
cubic  centimetre  (Kitasato).  Pigeons  are  very  slightly  susceptible. 
The  tetanic  symptoms  are  first  developed  in  the  vicinity  of  the  point 
of  inoculation  ;  if  the  animal  is  inoculated  in  the  posterior  portion  of 
the  body  the  hind  legs  first  show  tetanic  contraction,  if  in  the  fore 
part  of  the  body  the  muscles  of  the  neck  are  first  affected.  At  the 
autopsy  there  is  a  certain  amount  of  hyperaemia  at  the  point  of  in- 
oculation, but  no  pus  is  formed  ;  in  inoculations  with  garden  earth, 
or  accidental  inoculations  in  man,  pus  is  commonly  found  in  the 
vicinity  of  the  inoculation  wound.  The  various  organs  are  normal 
in  appearance.  Kitasato  says  that  he  has  not  been  able  to  demon- 
strate the  presence  of  the  bacillus  or  of  spores  in  the  spinal  marrow, 
the  nerves,  muscles,  spleen,  liver,  lungs,  kidneys,  or  blood  from  the 
heart ;  nor  has  he  been  able  to  obtain  cultures  from  the  various 
organs.  In  mice  which  were  inoculated  at  the  root  of  the  tail 
Kitasato  was  able  to  demonstrate  the  presence  of  the  bacilli  at 
the  point  of  inoculation  by  the  microscopical  examination  of  an 
excised  piece  of  the  tissues  for  eight  to  ten  hours  after  the  inocula- 
tion ;  later  than  this  they  were  not  found.  In  pus  from  the  inocu- 
lation wounds  of  men  and  animals  accidentally  infected  the  bacilli 
are  present,  but  the  formation  of  spores  does  not  always  oc- 
cur. According  to  Kitasato,  the  sooner  death  has  occurred  after 
accidental  inoculation  the  less  likely  are  spores  to  be  found  in  the 
rods,  but  from  pus  in  which  no  spores  are  seen  cultures  of  the 
bacillus  may  be  obtained  in  which  spores  will  develop  in  the  usual 
manner. 

Guinea-pigs  are  even  more  susceptible  to  the  tetanus  poison  than 
mice,  and  rabbits  less  so.  The  amount  of  filtrate  from  a  slightly 
alkaline  bouillon  culture  required  to  kill  a  mouse  is  extremely  minute 
— 0.00001  cubic  centimetre  (Kitasato).  The  tetanic  symptoms  are  de- 
veloped within  three  days  ;  if  the  animal  is  not  affected  within  four 
days  it  escapes  entirely.  The  tetanus  poison  is  destroyed  by  a  tem- 
perature of  65°  C.  maintained  for  five  minutes,  or  60°  for  twenty 
minutes,  or  55°  for  an  hour  and  a  half  ;  in  the  incubating  oven  at 
37°  C.  it  gradually  loses  its  toxic  potency ;  in  diffuse  daylight,  also, 
its  toxic  power  is  gradually  lost ;  in  a  cool,  dark  place  it  retains  its 
original  potency  indefinitely  ;  in  direct  sunlight  it  is  completely  de- 
stroyed in  from  fifteen  to  eighteen  hours  ;  it  is  not  injured  by  being 
largely  diluted  with  distilled  water ;  it  is  destroyed  in  an  hour  by 
hydrochloric  acid  in  the  proportion  of  0.55  per  cent ;  terchloride  of 
iodine  destroys  it  in  the  proportion  of  0. 5  per  cent,  cresol  in  1  per 
cent — one  hour's  exposure.  In  general  it  is  destroyed  by  acids  and 
by  alkalies.  Blood  serum  from  cattle,  horses,  sheep,  rabbits,  rats,  or 
guinea-pigs  does  not  modify  its  toxic  properties. 


536  PATHOGENIC   ANAEROBIC  BACILLI. 

The  researches  of  Tizzoni  and  Cattani  show  that  tetanus  spores 
preserved  upon  silk  threads  become  attenuated  after  a  time  when 
preserved  in  a  dark  place  in  free  contact  with  the  air.  Very  viru- 
lent cultures  liquefy  gelatin,  give  off  a  very  disagreeable  odor, 
and  have  a  decidedly  alkaline  reaction.  Less  virulent  cultures 
quickly  acquire  an  acid  reaction.  Cultures  of  which  the  virulence 
is  very  much  attenuated  grow  more  rapidly  and  abundantly  than 
virulent  cultures  and  produce  more  gas — in  hydrogen  at  37°  C. ;  they 
do  not  liquefy  gelatin  and  have  no  odor.  In  attenuated  cultures  de- 
generation forms  are  often  seen,  and  the  spores  are  frequently  elon- 
gated or  almost  rod-shaped.  Cultures  preserved  in  various  gases 
for  thirteen  to  fourteen  months  invariably  become  attenuated. 

Immunity. — Kitasato  was  not  able  to  produce  immunity  in  mice 
by  inoculations  with  minute  doses  of  the  poison,  or  with  a  filtrate 
which  had  been  exposed  to  various  degrees  of  temperature  by  which 
its  activity  was  diminished  or  destroyed.  But  immunity  lasting  for 
about  two  months  was  produced  in  rabbits  by  inoculating  them 
with  the  filtrate  from  a  culture  of  the  tetanus  bacillus  and  subse- 
quently, in  the  same  locality,  with  three  cubic  centimetres  of  a  one- 
per-cent  solution  of  terchloride  of  iodine  ;  this  last  solution  was  in- 
jected subcutaneously  in  the  same  dose  at  intervals  of  twenty-four 
hours  for  five  days.  Of  fifteen  rabbits  treated  in  this  way  six  proved 
to  be  immune  against  large  doses  of  a  virulent  culture  of  the  tetanus 
bacillus.  The  same  treatment  was  not  successful  in  producing  im- 
munity in  mice  or  guinea-pigs,  but  the  important  discovery  was 
made  that  a  small  quantity  of  blood  (0. 2  cubic  centimetre)  from  an 
immune  rabbit,  when  injected  into  the  abdominal  cavity  of  a  mouse, 
gave  it  immunity  from  the  effects  of  inoculations  with  the  tetanus 
bacillus.  Moreover,  mice  which  were  first  inoculated  with  a  virulent 
culture  of  the  bacillus,  and,  after  tetanic  symptoms  had  appeared,  re- 
ceived in  the  cavity  of  the  abdomen  an  injection  of  blood  serum  from 
an  immune  mouse,  were  preserved  from  death.  The  power  of  the 
blood  of  an  immune  animal  to  neutralize  the  tetanus  poison  was  fur- 
ther shown  by  mixing  the  filtrate  from  a  virulent  culture  with  blood 
serum  from  an  immune  animal  and  allowing  it  to  stand  for  twenty- 
four  hours ;  a  dose  three  hundred  times  greater  than  would  have 
sufficed  to  kill  a  mouse  proved  to  be  without  effect  after  such  admix- 
ture with  blood  serum— as  before  stated,  the  blood  serum  of  animals 
which  are  not  immune  has  no  effect  upon  the  poison.  The  duration 
of  immunity  induced  in  this  way  was  from  forty  to  fifty  days. 
Blood  serum  from  an  immune  rabbit,  preserved  in  a  cool,  dark  room, 
retains  its  power  of  neutralizing  the  tetanus  poison  for  about  a  week, 
after  which  time  it  gradually  loses  it.  Having  found  that  chickens 
have  a  natural  immunity  against  tetanus,  Kitasato  made  experiments 


PATHOGENIC   ANAEROBIC   BACILLI.  537 

to  ascertain  whether  their  blood  serum  would  also  neutralize  the 
tetanus  poison;  the  result  was  negative. 

That  the  tetanus  poison  is  present  in  the  blood  of  individuals  who 
die  from  tetanus  has  been  proved  by  Kitasato  by  injecting  a  small 
quantity  (0. 2  to  0. 3  cubic  centimetre)  of  blood  from  the  heart  of  a 
fresh  cadaver  into  mice;  the  animals  develop  typical  tetanic  symp- 
toms and  die  in  from  twenty  hours  to  three  days. 

Tizzoni  and  Cattani  have  (1891)  reported  results  similar  to  those 
obtained  by  Kitasato.  By  repeated  inoculations  with  gradually 
increasing  doses  of  the  tetanus  poison  they  succeeded  in  making 
a  dog  and  two  pigeons  immune,  and  found  that  blood  serum  from 
this  immune  dog,  in  very  small  amount,  completely  destroyed 
the  toxic  power  of  a  filtrate  from  cultures  of  the  tetanus  bacillus — 
one  to  two  drops  of  serum  neutralized  0. 5  cubic  centimetre  of  filtrate 
after  fifteen  to  twenty  minutes'  contact.  They  also  ascertained  that 
small  amounts  of  blood  serum  from  this  immune  dog  injected  into 
other  dogs  or  white  mice  produced  immunity  in  these  animals  ;  but 
they  were  not  able  to  produce  immunity  in  guinea-pigs  or  rabbits  by 
the  same  method. 

In  a  later  communication  (May,  1891)  Tizzoni  and  Cattani  give 
an  account  of  their  experiments  made  with  a  view  to  determining 
the  nature  of  the  substance  in  the  blood  serum  of  an  immune  animal 
which  has  the  power  of  destroying  the  toxalbumin  of  tetanus — "  tet- 
anus antitoxin/'  They  found,  in  the  first  place,  that  this  antitoxin 
in  blood  serum  is  destroyed  in  half  an  hour  by  a  temperature  of  68° 
C. ;  further,  that  it  does  not  pass  through  a  dialyzing  membrane ; 
that  it  is  destroyed  by  acids  and  alkalies.  As  a  result  of  their  re- 
searches they  conclude  that  it  is  an  albuminous  substance  having  the 
nature  of  an  enzyme. 

Vaillard  has  succeeded  in  producing  immunity  in  rabbits  by  re- 
peated injections  into  the  circulation  of  filtered  cultures — in  all 
twenty  cubic  centimetres — which  had  been  exposed  for  one  hour  to 
a  temperature  of  60°  C.  At  a  temperature  of  65°  C.  both  the  toxic 
and  the  immunizing  action  is  destroyed. 

186.    BACILLUS   CEDEMATIS   MALIGNI. 

Synonyms. — Bacillus  of  malignant  oedema ;  Vibrion  septique 
(Pasteur). 

Discovered  by  Pasteur  (1877);  carefully  studied  by  Koch  (1881). 
This  bacillus  is  widely  distributed,  being  found  in  the  superficial 
layers  of  the  soil,  in  dust,  in  putrefying  substances,  in  the  blood  of 
animals  which  have  been  suffocated  (by  invasion  from  the  intestine), 
in  foul  water,  etc. 

It  may  usually  be  obtained  by  introducing  beneath  the  skin  of  a 
38 


538 


PATHOGENIC   ANAEROBIC   BACILLI. 


rabbit  or  a  guinea-pig  a  small  quantity  of  garden  earth.  The  animal 
dies  within  a  day  or  two,  and  this  bacillus  is  found  in  the  bloody 
serum  effused  in  the  subcutaneous  connective  tissue  for  a  consider- 
able distance  about  the  point  of  inoculation. 

Morphology.—  Bacilli  from  3  to  3.5  p  long  and  1  to  1.1  j*  broad; 


Fio.   164.— Bacillus  oedematis  inaligni,  from  subcutaneous  connective  tissue  of  inoculated 
guinea-pig.    X  960.    (Baumgarten.) 


oo    I 


frequently  united  in  pairs,  or  chains  of  three  elements ;  may  grow 
out  into  long  filaments  15  to  40  //  long — these  are  straight,  or  bent 
at  an  angle,  or  more  or  less  curved.  They  resemble  the  bacillus  of 

anthrax,  but  are  not  quite  as  broad,  have 
rounded  ends,  and  in  stained  preparations 
the  long  filaments  are  not  segmented  as  is 
the  case  with  the  anthrax  bacillus.  By 
Loffler's  method  of  staining  they  are  seen  to 
have  flagella  arranged  around  the  periphery 
of  the  cells.  Large,  oval  spores  may  be  de- 
veloped in  the  bacilli  (not  in  the  long  fila- 
ments), which  are  of  greater  diameter  than 
the  rods,  and  produce  a  terminal  or  central 
swelling  of  the  same,  according  to  the  loca- 
tion of  the  spore. 

Stains  readily  by  the  aniline  colors  usu- 
ally employed,  but  is  decolorized  when  treated  by  Gram's  method. 
In  stained  preparations  the  long  filaments  may  present  a  somewhat 
granular  appearance  from  unequal  action  of  the  staining  agent. 

Biological  Characters. — A  strictly  anaerobic,  liquefying,  mo- 
tile bacillus.     Forms  spores.      Grows  in  the  usual  culture  media 


Fio.  165 — Bacillus  cedema- 
tis  malign! .  from  an  agar  cul- 
ture, showing  spores,  x  1,000 
From  a  photomicrograph. 
(Frfmkel  and  Pfeiffer.) 


PATHOGENIC  ANAEROBIC  BACILLI. 


539 


when  oxygen  is  excluded — in  an  atmosphere  of  hydrogen.  Grows 
at  the  room  temperature — better  in  the  incubating  oven  at  37°  C. 
The  spores  are  formed  most  abundantly  in  cultures  kept  in  the  in- 
cubating oven,  but  may  also  be  formed  at  a  temperature  of  20°  C. 
In  the  bodies  of  animals  which  succumb  to  an  experimental  inocula- 
tion no  spores  are  found  immediately 
after  death,  but  the  bacilli  multiply  rap- 
idly in  the  cadaver,  and  form  spores 
when  the  temperature  is  favorable. 

The  malignant- oedema  bacillus  may 
be  cultivated  in  ordinary  nutrient  gela- 
tin, but  its  development  is  more  abun- 
dant when  one  to  two  per  cent  of  grape 
sugar  has  been  added  to  the  culture 
medium.  In  deep  stick  cultures  in  this 
medium  development  occurs  at  first  only 
near  the  bottom  of  the  line  of  puncture  ; 
the  gelatin  is  liquefied  and  has  a  grayish- 
white,  clouded  appearance  ;  an  abundant 
development  of  gas  occurs,  and  as  this 
accumulates  the  growth  and  liquefaction 
of  the  gelatin  extend  upward.  A  very 
characteristic  appearance  is  obtained 
when  the  bacilli  are  mixed  in  a  test 
tube  with  gelatin  which  has  been  liquefied  by  heat,  and  which  is  then 
allowed  to  solidify.  Spherical  colonies  are  developed,  in  the  course 
of  two  or  three  days,  in  the  lower  portion  of  the  gelatin  ;  these  are 
filled  with  liquefied  gelatin  of  a  grayish- white  color,  and  when  ex- 
amined with  a  low  power  are  seen  to  be  permeated  with  a  network 
of  filaments,  while  the  periphery  presents  a  radiate  appearance.  In 
nutrient  agar  growth  also  occurs  at  the  bottom  of  a  deep  punc- 
ture ;  it  has  an  irregular,  jagged  outline  and  a  granular  appearance; 
the  considerable  development  at  the  deepest  portion  and  gradual 
thinning  out  above  give  the  growth  a  club  shape  ;  in  the  incubating 
oven  there  is  an  abundant  development  of  gas,  which  often  splits  up 
the  agar  medium  and  forces  the  upper  portion  against  the  cotton 
stopper.  An  abundant  development  of  gas  also  occurs  in  cultures 
in  blood  serum,  and  the  medium  is  rapidly  liquefied  ;  at  a  tempera- 
ture of  37°  it  is  changed  in  a  few  days  to  a  yellowish  fluid,  at  the 
bottom  of  which  some  irregular,  corroded  fragments  of  the  solidified 
serum  may  be  seen.  In  agar  plates,  placed  in  a  close  receptacle 
from  which  oxygen  is  excluded,  cloudy,  dull-white  colonies  are 
formed  which  have  irregular  outlines  and  under  the  microscope 
are  seen  to  be  made  up  of  branching  and  interlaced  filaments  radi- 


Fio.  166.— Bacillus  oedematis  ma- 
ligni,  cultures  in  nutrient  gelatin;  a, 
long  stick  culture;  6,  colonies  at  bot- 
tom of  gelatin  tube.  (Flugge.) 


540  PATHOGENIC   ANAEROBIC   BACILLI. 

ating  from  the  centre.  Cultures  of  the  malignant-oedema  bacillus 
give  off  a  peculiar,  disagreeable  odor,  which  cannot,  however,  be 
designated  as  "  putrefactive." 

Pathogenesis. — Pathogenic  for  mice,  guinea-pigs,  rabbits,  and, 
according  to  Kitt,  for  horses,  dogs,  goats,  sheep,  calves,  pigs,  chick- 
ens, and  pigeons.  According  to  Arloing  and  to  Chauveau,  cattle  are 
immune.  The  disease  is  rarely  developed  except  as  a  result  of  ex- 
perimental inoculations,  but  horses  occasionally  have  malignant 
oedema  from  accidental  inoculation,  and  cases  have  been  reported 
in  man — "gangrene  gazeuse."  A  small  quantity  of  a  pure  cul- 
ture injected  beneath  the  skin  of  a  susceptible  animal  gives  rise  to 
an  extensive  inflammatory  cedema  of  the  subcutaneous  connective 
tissue  and  of  the  superficial  muscles,  which  extends  from  the  point 
of  inoculation,  especially  towards  the  more  dependent  portions  of 
the  body.  The  bloody  serum  effused  is  without  odor  and  contains 
little  if  any  gas.  But  when  malignant  cedama  results  from  the  in- 
troduction of  a  little  garden  earth  beneath  the  skin  of  a  guinea-pig  or 
other  susceptible  annual,  the  effused  serum  is  frothy  and  has  a  pu- 
trefactive odor,  no  doubt  from  the  presence  of  associated  bacteria. 
Injections  into  the  circulation  do  not  give  rise  to  malignant  oedema, 
unless  at  the  same  time  some  bacilli  are  thrown  into  the  connective 
tissue.  While  small  animals  usually  die  from  an  experimental  in- 
oculation with  a  moderately  small  quantity  of  a  pure  culture,  larger 
ones  (dogs,  sheep)  frequently  recover.  At  the  autopsy,  if  made  at 
once,  the  bacilli  are  found  in  great  numbers  in  the  effused  serum, 
but  not  in  blood  from  the  heart  or  in  preparations  made  from  the 
parenchyma  of  the  various  organs  ;  later  they  may  be  found  in  all 
parts  of  the  body  as  a  result  of  post-mortem  multiplication.  This 
applies  to  rabbits  and  to  guinea-pigs,  but  not  to  mice  ;  in  these  little 
animals  the  bacilli  may  find  their  way  into  the  blood  during  the  last 
hours  of  life,  and  their  presence  may  be  demonstrated  in  smear  prepa- 
rations of  blood  from  the  heart  or  from  the  parenchyma  of  the  spleen 
or  liver.  In  mice  the  spleen  is  considerably  enlarged,  dark  in  color, 
and  softened ;  in  rabbits  and  guinea-pigs  less  so.  With  this  excep- 
tion the  internal  organs  present  no  very  notable  pathological  changes. 

Animals  which  recover  from  malignant  cedema  are  said  to  be 
subsequently  immune  (Arloing  and  Chauveau).  Boux  and  Cham- 
berlain have  shown  that  immunity  may  be  induced  in  guinea-pigs  by 
injecting  filtered  cultures  of  the  malignant-oedema  bacillus  (about 
one  hundred  cubic  centimetres  of  a  bouillon  culture  in  three  doses) 
into  the  abdominal  cavity  ;  or,  better  still,  by  the  injection  of  fil- 
tered serum  from  animals  which  have  recently  succumbed  to  an  ex- 
perimental inoculation  (one  cubic  centimetre  repeated  daily  for 
even  or  eight  days). 


PATHOGENIC   ANAEROBIC   BACILLI. 


541 


187.   BACILLUS  CADAVERIS. 

Obtained  by  the  writer  (1889)  from  pieces  of  liver  and  kidney,  from  yel- 
low-fever cadavers,  which  had  been  preserved  for  forty-eight  hours  in  an 
antiseptic  wrapping,  at  the  summer  temperature  of  Havana;  also  in  two 


FIG.  167.— Bacillus  cadaveris;  smear  preparation  from  liver  of  yellow-fever  cadaver,  kept 
twenty-four  hours  in  an  antiseptic  wrapping,    x  1,000.    From  a  photomicrograph.    (Sternberg.) 

cases  from  pieces  of  yellow-fever  liver  immediately  after  the  autopsy ;  also 
from  liver  preserved  in  an  antiseptic  wrapping  from  comparative  autopsies 
made  in  Baltimore. 

Morphology. — Large  bacilli  with  square  or  slightly  rounded  corners, 
from  1.5  to  4  i*  in  length  and  about  1.2  u.  broad;  frequently  associated  in 
pairs ;  may  grow  out  into  straight  or 
slightly  curved  filaments  of  from  5 
to  15  >u  in  length. 

Biological  Characters.— AIL  an- 
aerobic, non-motile  bacillus;  not 
cultivated  in  nutrient  gelatin;  not 
observed  to  form  spores. 

Bacillus  cadaveris  is  a  strict  anae- 
robic and  is  difficult  to  cultivate.  I 
have  succeeded  best  with  nutrient 
agar  containing  five  per  cent  of 
glycerin,  removing  the  oxygen 
thoroughly  by  passing  a  stream  of 
hydrogen  through  the  liquefied  me- 
dium. The  colonies  in  a  glycerin- 
agar  roll  tube  (containing  hydrogen 
and  hermetically  sealed)  are  opaque, 
irregular  in  outline,  granular,  and  of 
a  white  color  by  reflected  light. 
The  culture  medium  acquires  an 
acid  reaction  as  a  result  of  the  de- 
velopment of  the  bacillus. 

Liver  tissue  containing-  this  bacillus,  after  having  been  kept  in  an  anti- 
septic wrapping  for  forty-eight  hours,  has  a  fresh  appearance,  a  very  acid  re- 
action, and  is  without  any  putrefactive  odor. 


FIG.  168.— Bacillus  cadaveris,  from  an  anae- 
robic culture  in  glycerin-agar.  x  1,000.  From 
a  photomicrograph.  (Sternberg.) 


542 


PATHOGENIC   ANAEROBIC   BACILLI. 


tissue  containing  this  bacillus  is  very  pathogenic 
for  guinea-pigs  when  injected  subcutaneously,  and  causes  an  extensive  in- 
flammatory oedema  extending  from  the  point  of  inoculation.  Pure  cul- 
tures of  the  bacillus  are  less  pathogenic,  and  the  few  experiments  which  I 
made  in  Havana  gave  a  somewhat  contradictory  result,  recovery  having 
occurred  in  one  guinea-pig  which  received  a  subcutaneous  injection  of  ten 
minims  of  liquid  from  an  anaerobic  culture  in  glycerin-agar,  while  another 
died  at  the  end  of  twenty  hours  from  a  subcutaneous  injection  of  three 
minims,  with  extensive  inflammatory  oedema  in  the  vicinity  of  the  point  of 
inoculation. 


188.    BACILLUS   OF   SYMPTOMATIC   ANTHRAX. 

Synonyms. — Rauschbrandbacillus,  Ger. ;  Bacille  du  charbon 
symptomatique,  Fr. 

First  described  by  Bellinger  and  Feser  (1878);  carefully  studied 
and  its  principal  characters  determined  by  Arloing,  Cornevin,  and 
Thomas  (1880-83). 


FIG.  169.  Fio.  170. 

FIG.  169.— Bacillus  of  symptomatic  anthrax,  from  an  agar  culture,  x  1,000.  From  a  photomi- 
crograph. (FrSnkel  and  Pfeiffer.) 

FIG.  170.— Bacillus  of  symptomatic  anthrax,  from  muscles  of  inoculated  guinea-pig.  From  a 
photomicrograph.  (Roux.) 

Found  in  the  affected  tissues  of  animals— principally  cattle— suf- 
fering from  ' '  black  leg, "  ' '  quarter  evil, "  or  symptomatic  anthrax  (Fr. , 
"charbon  symptomatique";  Ger.,  "Rauschbrand").  The  disease 
prevails  during  the  summer  months  in  various  parts  of  Europe,  and 
is  characterized  by  the  appearance  of  irregular,  emphysematous 
swellings  of  the  subcutaneous  tissue  and  muscles,  especially  over  the 
quarters,  hence  the  name  "quarter  evil."  The  muscles  in  the 


PATHOGENIC   ANAEROBIC   BACILLI. 


543 


affected  areas  have   a  dark  color  and  contain  a  bloody  serum  in 
which  the  bacillus  is  found. 

Morphology. — Bacilli  with  rounded  ends,  from  three  to  five  /* 
long  and  0.5  to  0.6  /*  broad  ;  sometimes  united  in  pairs,  but  do  not 
grow  out  into  filaments.  The  spores  are  oval,  somewhat  flattened  on 
one  side,  thicker  than  the  bacilli,  and  lie  near  the  middle  of  the  rods, 
but  a  little  nearer  to  one  extremity.  The  bacilli  containing  spores 
are  somewhat  spindle-formed  (Kitasato).  "Involution  forms"  are 
quite  common  in  old  cultures  or  in  unfavorable 
media ;  in  such  cultures  variously  distorted  and 
often  greatly  enlarged  bacilli  may  be  seen,  some 
being  greatly  swollen  in  the  middle  —  spindle- 
shaped.  When  properly  stained,  by  Loffler's 
method,  a  number  of  flagella  are  seen  around  the 
periphery  of  the  cells. 

Stains  with  the  aniline  colors  usually  em- 
ployed, but  not  by  Gram's  method.  Spore-bear- 
ing bacilli  may  be  double-stained  by  first  stain- 
ing the  spores  by  ZiehTs  method,  and  then  the 
bacilli  with  a  solution  of  methylene  blue. 

Biological  Characters. — An  anaerobic,  liq- 
uefying, mot i '1 e  bacillus.  Forms  spores.  Grows 
at  the  room  temperature  in  the  usual  culture  media, 
in  the  absence  of  oxygen,  in  an  atmosphere  of  hy- 
drogen, but  not  in  carbon  dioxide.  This  bacillus 
grows  more  rapidly  and  abundantly  in  nutrient 
agar  or  gelatin  to  which  1.5  to  2  per  cent  of 
grape  sugar  or  five  per  cent  of  glycerin  has  been 
added.  Colonies  in  gelatin,  in  an  atmosphere  of 
hydrogen,  are  at  first  spherical,  with  irregular  out- 
lines and  a  wart-like  surface  ;  later  the  gelatin  is 
liquefied  around  them,  and  radiating  filaments 
grow  out  into  the  gelatin,  so  that  by  transmitted 
light  they  present  the  appearance  of  an  opaque 
central  mass  with  an  irregular  surface  surrounded 
by  rays.  In  stick  cultures  in  nutrient  gelatin,  at 
20°  to  25°  C.,  at  the  end  of  two  or  three  days 
development  occurs  at  the  bottom  of  the  line  of  puncture  to  within 
about  two  fingers'  breadth  of  the  surface  ;  the  gelatin  is  slowly 
liquefied  and  considerable  gas  is  formed.  In  old  cultures  the 
growth  and  liquefaction  of  the  gelatin  extend  nearly  to  the  sur- 
face. In  agar  stick  cultures,  in  the  incubating  oven,  develop- 
ment begins  within  a  day  or  two  and  extends  to  within  one 
finger's  breadth  of  the  surface ;  considerable  gas  is  evolved,  and 


FIG.  in.  —  Bacillus 
of  symptomatic  an- 
thrax; lon^  stick  cul- 
ture in  nutrient  gela- 
tin, ten  days  at  18°- 
90°  C.  CKitasato.) 


544  PATHOGENIC   ANAEROBIC   BACILLI. 

the  cultures  have  a  peculiar,  acid,  penetrating  odor.  Development 
is  most  rapid  at  36°  to  38°  C.,  but  may  occur  at  a  temperature  of  16° 
to  18°  C. — not  lower  than  14°.  Spores  are  quickly  formed  in  cul- 
tures kept  in  the  incubating  oven — not  so  quickly  at  the  room  tem- 
perature. These  withstand  a  temperature  of  80°  C.  maintained  for 
an  hour,  but  are  killed  in  five  minutes  by  a  temperature  of  100°  C. 
(in  steam).  In  the  bodies  of  infected  animals  spores  are  not  formed 
until  after  the  death  of  the  animal,  at  the  end  of  twenty-four  to  forty- 
eight  hours  (Kitasato). 

The  spores  are  destroyed  by  a  five-per-cent  solution  of  carbolic 
acid  in  ten  hours,  and  the  bacilli,  in  the  absence  of  spores,  in  five 
minutes  ;  a  1 : 1,000  solution  of  mercuric  chloride  destroys  the  spores 
in  two  hours  (Kitasato).  According  to  Kitasato,  certain  shining 
bodies  of  irregular  form,  which  stain  readily  with  the  aniline  colors, 
are  to  be  seen  in  the  rods  as  they  are  found  in  the  bloody  serum  from 
an  animal  recently  dead  ;  but  these  are  not  spores,  as  some  bacterio- 
logists have  supposed. 

Pathogenesis. — Cattle,  which  are  immune  against  malignant 
oedema,  are  most  subject  to  infection  by  the  bacillus  of  symptomatic 
anthrax,  and  the  disease  produced  by  this  anaerobic  bacillus  prevails 
almost  entirely  among  them  ;  horses  are  not  attacked  spontaneously 
— i.e.,  by  accidental  infection — and  when  inoculated  with  a  culture  of 
this  bacillus  present  only  a  limited  local  reaction.  Swine,  dogs,  rab- 
bits, fowls,  and  pigeons  have  but  slight  susceptibility,  but  the  re- 
searches of  Arloing,  Cornevin,  and  Thomas,  and  of  Roger  show  that 
by  the  addition  of  a  twenty-per-cent  solution  of  lactic  acid  to  a  cul- 
ture its  virulence  is  greatly  increased,  and  animals  which  have  but 
little  susceptibility,  like  the  rabbit  or  the  mouse,  succumb  to  such  in- 
jections ;  similar  results  were  obtained  by  Roger  by  the  simultaneous 
injection  of  sterilized  or  non-sterilized  cultures  of  Bacillus  prodigiosus 
or  of  Proteus  vulgaris.  The  guinea-pig  is  the  most  susceptible  ani- 
mal. When  inoculated  subcutaneously  with  a  small  quantity  of  a 
pure  culture,  or  with  spores  attached  to  a  silk  thread,  it  dies  in  from 
twenty-four  to  thirty-six  hours.  At  the  autopsy  a  bloody  serum  is 
found  in  the  subcutaneous  tissues  in  the  vicinity  of  the  point  of  in- 
oculation, and  the  muscles  present  a  dark-red  or  black  appearance 
similar  to  that  in  cattle  affected  with  "  black  leg."  The  internal  or- 
gans present  no  notable  pathological  changes.  Immediately  after 
death  the  bacilli  are  found  only  in  the  effused  serum  and  the  affected 
tissues  near  the  point  of  inoculation,  but  later  they  multiply  in  the 
cadaver  and  are  found  throughout  the  body.  According  to  Kitasato, 
the  cultures  in  solid  media  preserve  their  virulence  for  an  indefinite 
period,  but  cultures  in  a  bouillon  made  from  the  flesh  of  guinea-pigs 
soon  lose  their  virulence.  Cultures  are  readily  attenuated  by  heat 


PATHOGENIC   ANAEROBIC   BACILLI.  545 

according  to  the  method  of  Toussaint  and  Chauveau  ;  a  temperature 
of  42°  to  43°  C.  is  suitable.  The  pathogenic  virulence  of  spores  may 
also  be  attenuated  by  subjecting  them  to  dry  heat — a  temperature  of 
80°  to  100°  C.  maintained  for  several  hours.  For  the  production  of 
immunity  in  cattle  Arloing,  Cornevin,  and  Thomas  recommend  the 
use  of  a  dried  powder  of  the  muscles  of  animals  which  have  suc- 
cumbed to  the  disease,  and  which  has  been  subjected  to  a  suitable 
temperature  to  insure  attenuation  of  the  pathogenic  virulence  of  the 
spores  contained  in  it.  Kitt,  who  has  made  extended  experiments 
with  this  bacillus,  recommends  that  the  muscles  be  first  dried  at  32° 
to  35°  C.  and  then  powdered.  Two  vaccines  are  then  prepared — a 
stronger  vaccine  by  exposure  of  a  portion  of  the  powder  to  a  tem- 
perature of  85°  to  90°  C.  for  six  hours,  and  a  weaker  vaccine  by  ex- 
posure for  six  hours  to  a  temperature  of  100°  to  104°  C.  (dry  heat), 
Inoculations  made  with  this  attenuated  virus — the  weakest  first  and 
subsequently  the  least  attenuated — give  rise  to  a  local  reaction  of 
moderate  intensity,  and  the  animal  is  subsequently  immune  from  the 
effects  of  the  most  virulent  material.  Immunity  may  also  be  secured 
by  intravenous  inoculations  ;  or,  in  guinea-pigs,  by  inoculations  with 
bouillon  cultures  which  have  been  kept  for  a  few  days  and  as  a  re- 
sult have  lost  their  original  virulence,  or  with  cultures  kept  in  an  in- 
cubating oven  at  a  temperature  of  42°  to  43°  C. ;  or  by  inoculation 
with  a  very  minute  quantity  of  a  pure  culture  ;  or  by  an  inoculation 
made  into  the  extremity  of  the  tail ;  or  by  inoculations  with  filtered 
cultures  (Roux  and  Chamberlain),  or  with  cultures  sterilized  by  heat 
(Kitasato).  It  has  been  claimed  (Roux)  that  animals  which  have 
been  made  immune  against  symptomatic  anthrax  are  also  immune 
against  malignant  oedema.  But  in  a  carefully  conducted  series  of 
experiments  Kitasato  was  unable  to  confirm  this  ;  he  found  that 
guinea-pigs  which  had  an  immunity  against  the  most  virulent  cul- 
tures of  the  Rauschbrand  bacillus  succumbed  invariably  to  malig- 
nant oedema  when  inoculated  subcutaneously  with  the  bacillus  of 
malignant  oedema. 

Klein  (1894)  has  obtained  from  the  spleen  of  sheep  a  bacillus 
which  corresponds  with  the  bacillus  of  malignant  oedema  in  every 
respect,  except  that  it  proved  to  be  without  pathogenic  power — "a 
non-virulent  variety  of  the  Rauschbrand  bacillus"  (Klein). 

189.    BACILLUS   CEDEMATIS  MALIGNI   NO.  II.    (Novy). 

Obtained  by  Novy  (1894)  from  the  subcutaneous  oedema  in  guinea-pigs 
which  were  inoculated  with  a  solution  of  milk-nuclein,  which  had  been  pre- 
pared from  fresh  casein. 

Morphology. — Bacilli  with  rounded  ends,  usually  solitary,  from  2.5  to 
5  fj-  long  and  from  0.8  to  0.9  //  broad.  Occasionally  short  and  straight  fila- 
ments, 8  to  14  //  long,  are  seen — very  rarely  these  reach  a  length  of  22  to  35  n. 


546  PATHOGENIC   ANAEROBIC   BACILLI. 

Long  and  slender  spiral  filaments  are  found  in  pure  cultures  which  are  be- 
lieved to  be  gigantic  flagella.  These  are  seen  in  preparations  stained  with 
gentian  violet  as  unstained  spiral  filaments,  usually  from  17  to  25  n  long ; 
some  are  of  uniform  thickness  and  others  spindle-formed,  having  a  thickness 
of  1.7  to  2.6  p  in  the  middle,  and  tapering  to  a  scarcely  visible  line  at  the  ex- 
tremities. These  flagella  are  readily  stained  by  Loffler's  method.  They  are 
attached  to  the  periphery  of  the  rods,  as  in  the  typhoid  bacillus.  In  artifi- 
cial cultures  they  are  usually  from  40  to  50  /"  long.  With  reference  to  the 
peculiar  spindle-formed  bodies  found  in  the  cultures  Novy  says  :  "  As  to  the 
character  of  these  gigantic  flagella  little  can  be  said.  Loftier,  who,  so  far  as 
I  know,  was  the  first  to  observe  these  singular  forms,  regarded  them  as  bun- 
dles or  collections  of  flagella." 

Although  at  first  inclined  to  doubt  this,  Novy  says,  in  a  postscript  to  his 
paper,  that  an  examination  of  photo-micrographs,  which  had  been  made  to 
accompany  it,  convinces  him  that  Loffler's  explanation  is  probably  correct. 

Biological  Characters. — An  anaerobic,  motile  bacillus.  The  motions  are 
not  active,  but  consist  in  a  very  moderate  to-and-fro  swinging  motion.  Does 
not  form  spores.  Does  not  grow  at  the  room  temperature.  Grows  at  tem- 
peratures of  24°  to  38°  C.  The  best  media  for  its  development  are  slightly 
alkaline  bouillon,  gelatin,  or  agar,  containing  two  per  cent  of  glucose.  May 
be  cultivated  in  a  vacuum  or  in  an  atmosphere  of  hydrogen,  carbon  dioxide, 
or  illuminating  gas.  Also  in  long  stick  cultures  in  agar.  In  glucose-agar 
plates  colonies  develop  in  fifteen  hours  at  38  C.  These  appear  as  small, 
white  masses  the  size  of  a  pin's  head,  which,  under  the  microscope,  appear 
to  be  made  up  of  thickly  felted  threads.  The  smaller  colonies  appear  as  a 
network  of  branching  lires,  very  similar  to  the  colonies  of  the  tetanus  ba- 
cillus ;  larger  colonies  have  a  dark  centre,  with  an  irregular,  fringed  margin, 
and  are  surrounded  by  delicate  filaments.  In  glucose-agar  stick  cultures 
growth  occurs  along  the  line  of  puncture  to  within  one  cubic  centimetre  of 
the  surface,  but  is  not  as  abundant  as  the  growth  of  the  bacillus  of  malig- 
nant oedema  or  of  symptomatic  anthrax.  At  38°  C.  development  occurs  within 
twelve  to  sixteen  hours,  and  has  reached  its  maximum  at  the  end  of  twenty- 
four  hours.  An  abundant  development  of  gas  occurs,  which  splits  up  the 
agar  and  forces  the  upper  portion  towards  the  top  of  the  tube.  The  develop- 
ment of  gas  is  most  abundant  in  alkaline  media,  being  almost  absent  in  media 
having  a  neutral  or  acid  reaction.  The  most  favorable  medium  is  a  fresh  al- 
kaline bouillon  containing  two  per  cent  of  gelatin,  of  glucose,  and  of  pep- 
tone. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  white  mice,  white  rats, 
pigeons,  and  cats.  Death  usually  results  in  from  twelve  to  thirty-six  hours 
after  the  subcutaneous  injection  of  one-tenth  to  one-fourth  cubic  centimetre 
of  a  pure  culture.  At  the  autopsy  an  extensive  subcutaneous  cedema  is  found 
extending  from  the  point  of  inoculation.  The  fluid  in  the  brawny  connective 
tissue  is  usually  colorless,  sometimes  of  a  pale-red  color.  A  small  amount  of 
gas  is  commonly  present.  The  pleura!  cavities  contain  an  enormous  amount 
of  serous  exudate,  which  at  first  is  fluid,  but  when  the  autopsy  is  delayed  be- 
comes gelatinous.  In  rabbits  and  guinea-pigs  the  amount  of  this  serum  ob- 
tained from  the  pleural  cavities  may  be  from  fifty  to  sixty  cubic  centime!  r» «s. 
The  bacilli  are  usually  not  very  numerous  in  this  serum  from  the  subcuta- 
neous tissues  and  pleural  cavity. 

Kerry  (1894)  has  described  a  "  new  pathogenic  anaerobic  bacillus  "  which 
resembles  that  of  Novy  in  several  particulars.  It  does  not  grow  at  the  ropin 
temperature,  does  not  form  spores,  and  is  pathogenic  for  mice,  rats,  rabbits, 
and  guinea-pigs  ;  it  forms  "very  long  and  thick  flagella,  which  may  be  sjn'r- 
alia  gechlangelt."  This  bacillus  was  obtained  from  a  guinea-pig  inoculated 
with  dried  blood  (suspended  in  water  containing  lactic  acid  and  glucose) 
which  had  been  obtained  from  a  cow  that  was  supposed  to  have  died  of 
Rauschbrand. 


PATHOGENIC    ANAEROBIC   BACILLI.  547 

190.    BACILLUS   PHLEGMONES   EMPHYSEMATOS^E    (E.    Frankel). 

Obtained  by  Frankel  (1893)  from  four  cases  of  "gas  phlegmon." 

Morphology. — Short,  thick  bacilli,  with  round  ends,  about  as  thick  as 
the  anthrax  bacillus,  usually  united  in  pairs ;  long  filaments  are  seen  in  gela- 
tin cultures,  and  in  the  tissues  of  infected  guinea-pigs. 

Biological  Characters. — An  anaerobic,  non-motile,  liquefying  bacillus. 
Spores  are  occasionally  seen  in  agar  cultures ;  these  are  spherical  and  lo- 
cated in  the  slightly  swollen  extremities  of  the  rods.  In  glucose-agar  (one 
per  cent  glucose)  plates,  in  an  atmosphere  of  hydrogen  at  37°  C.,  gas  bub- 
bles are  seen  upon  and  below  the  surface;  these  may  have  a  diameter  of 
one  centimetre,  and  small  bubbles  are  often  attached  to  the  larger  ones.  In 
other  places  the  agar  is  split  open  ;  in  others  still,  colonies  are  developed 
without  the  formation  of  gas  ;  these  are  round,  with  a  dark-brown  centre 
and  paler  margin.  In  stick  cultures  in  agar  containing  one  per  cent  of 
glucose  the  growth,  at  37°  C.,  is  abundant  at  the  end  of  twenty-four  hours 
all  along  the  line  of  puncture  ;  the  agar  is  split  up  by  gas,  and  bubbles  often 
accumulate  on  the  surf  ace.  In  gelatin  cultures,  in  an  atmosphere  of  hydro- 
gen at  the  end  of  two  or  three  days,  small,  round,  brownish-yellow,  slightly 
granular  colonies  are  developed,  which  later  appear  to  lie  in  an  air  bubble. 
In  gelatin  stick  cultures  growth  is  first  seen  one  to  two  centimetres  below  the 
surface  ;  after  several  days  spherical  colonies  are  developed  along  the  line 
of  puncture,  and  at  times  liquefaction  is  seen,  while  at  others  gas  bubbles 
are  developed.  Upon  blood  serum,  in  hydrogen,  an  abundant  development 
occurs  with  formation  of  gas  ;  these  cultures  give  off  a  fetid  odor.  In  milk 
coagulation  occurs,  but  no  gas  is  developed.  The  bacillus  dies  out  in  agar 
cultures  within  two  or  three  days,  unless  they  are  preserved  in  hydrogen. 
In  gelatin  cultures  it  survives  for  several  months. 

Pathogenesis. — In  guinea-pigs  subcutaneous  inoculation  gives  rise  to  the 
development  of  a  gas  phlegmon,  and  usually  to  the  death  of  the  animal. 
Not  pathogenic  for  mice  or  for  rabbits  when  injected  into  the  circulation. 

NOTES   RELATING   TO    THE    PREVIOUSLY   DESCRIBED   ANAEROBIC 

BACILLI. 

Tetanus  Bacillus.—  Brieger  and  Cohn,  in  investigations  (1893)  relating  to 
the  toxic  products  of  the  tetanus  bacillus,  have  arrived  at  the  following  re- 
sults :  The  cultures  were  made  in  veal  bouillon  containing  one  per  cent  of 
peptone  and  one- fifth  per  cent  of  chloride  of  sodium.  Large  quantities  of 
the  cultures  in  this  medium  were  filtered  through  porcelain  filters.  The 
active  substance  was  precipitated  from  the  filtrate  by  means  of  a  saturated 
solution  of  ammonium  sulphate.  By  adding  this  salt  in  excess  the  precipi- 
tate is  made  to  rise  to  the  surface  and  is  skimmed  off  with  a  platinum  spatula. 
The  liquid  is  removed  by  placing  this  upon  porous  porcelain  plates  and  the 
crude  toxin  is  dried  in  a  vacuum.  It  still  contains  6.5  per  cent  of  ammo- 
nium sulphate.  The  tetanus  bouillon  after  filtration  is  said  to  be  fatal  to 
mice  in  the  dose  of  0.00005  cubic  centimetre.  A  litre  of  this  bouillon  gave 
about  one  gramme  of  the  dried  precipitate,  which  produced  characteristic  tet- 
anic symptoms  and  death  when  injected  into  mice  in  the  dose  of  0.0000001 
gramme.  Kitasato,  in  his  experiments,  had  previously  obtained  a  tetanus 
bouillon  which  was  five  times  as  toxic  as  that  used  by  Brieger  and  Cohn  in 
their  experiments,  and  which  killed  mice  in  the  dose  of  0.00001  cubic  centi- 
metre. The  dried  precipitate  obtained  by  Brieger  and  Cohn  contained  vari- 
ous impurities,  including  a  certain  amount  of  ammonium  sulphate,  but  was 
found  to  kill  susceptible  animals  in  the  proportion  of  0.0000066  gramme  per 
kilogramme  of  body  weight. 

It  was  purified  without  loss  of  toxic  power  by  placing  it  in  a  dialyzer  in 
running  water  for  from  twenty-four  to  forty-eight  hours,  after  which  it  was 


548  PATHOGENIC   ANAEROBIC   BACILLI. 

dried  in  vacuo  at  20°  to  22°  C.  The  purified  toxin  as  thus  obtained  had  a 
slightly  yellowish  color,  and  was  in  the  form  of  transparent  scales,  which 
were  odorless,  tasted  like  gum  acacia,  and  were  easily  soluble  in  water.  The 
chemical  reactions  of  this  purified  toxin,  according  to  Brieger  and  Cohn,  show 
that  it  is  not  a  true  albuminous  body.  When  injected  beneath  the  skin  of  a 
mouse  weighing  fifteen  grammes,  in  the  dose  of  0.00000005  gramme,  it  causes 
its  death,  and  one-fifth  of  this  amount  gave  rise  to  tetanic  symptoms  from 
which  the  animal  recovered  after  a  time.  The  lethal  dose  for  a  man  weigh- 
ing seventy  kilogrammes  is  estimated  by  the  authors  named  to  be  0.00023 
gramme  (6.23  milligramme).  Comparing  this  with  the  most  deadly  vege- 
table alkaloids  known  it  is  nearly  six  hundred  times  as  potent  as  atropin  and 
one  hundred  and  fifty  times  as  potent  as  strychnin. 

Fermi  and  Pernossi  (1894),  as  a  result  of  an  elaborate  research,  have  deter- 
mined many  of  the  chemical  characters  of  the  tetanus  toxin.  "When  in  solu- 
tion it  is  destroyed  by  a  comparatively  low  temperature  (55°  C.  for  one  hour) 
and  by  exposure  to  direct  sunlight,  but  the  dry  powder  resists  a  temperature 
of  120°  C.  It  has  not  the  properties  of  an  alkaloid,  as  it  is  not  dissolved  by 
any  of  the  usual  solvents  of  these  bodies— the  only  solvent  thus  far  discovered 
is  said  to  be  water.  It  resembles  the  albumins  and  peptones  in  its  failure  to 
pass  through  a  dialyzing  membrane.  The  authors  last  referred  to  conclude 
their  summary  of  results  as  follows  : 

"The  appended  table  shows  that  the  tetanus  poison,  like  that  of  diphtheria, 
in  its  behavior  as  regards  the  action  of  light,  heat,  chemical  agents,  and 
dialysis,  as  also  its  solvents,  the  agents  which  precipitate  it,  and  its  action  upon 
living  animals,  closely  resembles  the  poisons  of  serpents  (Naja  tripudians, 
Crotalus,  etc.).  As  to  the  chemical  nature  of  this  group  of  substances,  we  can 
at  present  only  say  that  they  rather  have  the  characters  of  colloidal  sub- 
stances than  otherwise,  and  more  nearly  resemble  the  albuminoid  bodies  than 
the  bases.  We  do  not,  however,  reject  the  very  probable  hypothesis  that 
these  toxins  are  acids  or  bases,  or  other  very  unstable,  peculiar  substances 
which  are  closely  united  with  colloidal  substances,  as  is  the  case,  for  example, 
with  the  alkali  and  acid  albumins  and  so  many  other  albuminous  bodies." 

While  the  exact  nature  of  the  toxic  substance  contained  in  tetanus  cul- 
tures has  not  been  determined,  we  probably  cannot,  at  present,  do  better  than 
to  continue  to  speak  of  it  as  a  '*  toxalbumin." 

Symbiosis. — Car  bone  and  Perrero  (1895),  in  a  case  of  so-called  rheumatic 
tetanus,  in  which  there  was  no  evidence  of  a  wound  through  which  infection 
might  have  occurred,  obtained  the  tetanus  bacillus,  by  inoculations  in  mice, 
from  an  exudate  into  the  larger  bronchial  tubes.  The  micrococcus  of  pneu- 
monia was  also  present,  and  the  authors  named  report  that  as  a  result  of  asso- 
ciation with  this  coccus  the  tetanus  bacillus  is  able  to  grow  in  the  presence  of 
oxygen.  Other  bacteriologists  had  previously  reported  that  the  tetanus  bacil- 
lus is  able  to  grow  in  mixed  cultures  in  the  presence  of  oxygen,  and  this  has 
been  confirmed  by  the  recent  researches  of  Kedrowski  (1895).  Righi  ( 1 6 
claims  that  the  tetanus  bacillus  may  acquire  the  faculty  of  growing  in  the 
presence  of  oxygen,  when  it  is  gradually  habituated  to  the  presence  of  this 
gas.  Penzo  has  observed  a  similar  modification  in  the  biological  characters 
of  the  bacillus  of  malignant  oedema  ;  and  Kitt  (1895)  has  succeeded  in  obtain- 
ing aerobic  cultures  or  the  bacillus  of  symptomatic  anthrax.  He  says  :  ' '  The 
bouillon  cultures  do  not  always  develop  anaerobic ;  one  must  inoculate  sev- 
eral half-litre  flasks  and  place  them  in  the  incubating  oven  ;  some  remain 
clear  and  without  evidence  of  development,  however  long  they  are  kept ; 
others  begin  to  ferment  at  the  end  of  two  days.  Sometimes  this  formation  of 
gas  only  lasts  for  a  day  ;  again,  with  more  vigorous  development,  it  may  last 
for  several  days,  and  the  contents  of  the  flasks  have  the  appearance  of  bubbl  i  ii^r 
champagne  or  1 1 V  /.s.s-  bier.  When  a  culture  is  once  obtained  in  this  way  there 
is  no  difficulty  in  making  a  series  of  aerobic  cultures. 


STERNBERG'S  BACTERIOLOGY. 


PI., I  r-     \!|| 


Spirillum    Obonnoiori  in  blooil  of  two    inotilu-x  • 
niociilu  t  CM!  iifli-r   ri'iiM'v.il   ol    sleen. 


XV. 
PATHOGENIC  SPIRILLA. 

191.    SPIRILLUM  OBERMEIERI. 

Synonyms. — Spirochsete  Obermeieri  ;  Spirillum  of  relapsing  fe- 
ver ;  Die  Recurrensspirochate. 

Discovered  by  Obermeier  (1873)  in  the  blood  of  persons  suffering 
from  relapsing  fever. 

This  spirillum  is  present,  in  very  great  numbers,  in  the  blood  of 
relapsing-fever  patients  during  the  febrile  paroxysms.  It  has  not 
been  found  under  any  other  circumstances,  and  its  etiological  rela- 
tion to  the  disease  with  which  it  is  associated  is  generally  admitted. 

Morphology. — Very  slender,  flexible,  spiral  or  wavy  filaments, 
with  pointed  ends  ;  from  sixteen  to  forty  j*  in  length  and  consider- 
ably thinner  than  the  cholera  spirillum — about  0.1  /*.  Koch  has 
demonstrated  the  presence  of  flagella  (Eisenberg). 

Stains  readily  with  the  aniline  colors,  especially  with  fuchsin, 
Bismarck  brown,  and  in  Lofner's  solution  of  methylene  blue. 

Biological  Characters. — An  aerobic,  motile  spirillum  which 
has  not  been  cultivated  in  artificial  media.  This  spirillum  appears  to 
be  a  strict  parasite,  whose  habitat  is  the  blood  of  man.  The  disap- 
pearance of  the  parasite  from  the  blood  soon  after  the  termination 
of  a  febrile  paroxysm,  and  its  reappearance  during  subsequent  par- 
oxysms, have  led  to  the  inference  that  it  must  form  spores,  but  this 
has  not  been  demonstrated.  In  fresh  preparations  from  the  blood 
the  spirillum  exhibits  active  progressive  movements,  accompanied 
by  very  rapid  rotation  in  the  long  axis  of  the  spiral  filaments,  or  by 
undulatory  movements.  The  movements  are  so  vigorous  that  the 
comparatively  large  red  blood  corpuscles  are  seen,  under  the  micro- 
scope, to  be  thrown  about  by  the  slender  spiral  filaments,  which  it  is 
difficult  to  see  in  unstained  preparations.  When  preserved  in  a  one- 
half -per-cent  salt  solution  they  continue  to  exhibit  active  movements 
for  a  considerable  time.  Efforts  to  cultivate  this  spirillum  in  artificial 
media  have  thus  far  been  unsuccessful,  although  Koch  has  observed 
an  increase  in  the  length  of  the  spirilla  and  the  formation  of  a 
tangled  mass  of  filaments. 


550 


PATHOGENIC    SPIRILLA. 


In  experiments  made  by  Heydenreich  the  spirillum  was  found  to 
preserve  its  vitality  (motility)  for  fourteen  days  at  a  temperature  of 


Fio.    172.— Spirillum   Obermeieri  in  blood   of   man.    x   1,000.     From   a   photomicrograph. 
(FrSnkel  and  Pf  eiffer. ) 

16°  to  22°  0.,  for  twenty  hours  at  37°,  and  at  42.5°  for  two  or  three 
hours  only. 

Pathogenesis. — Causes  in  man  the  disease  known  as  relapsing 
fever.     Munch  and  Moczutkowsky  have  produced  typical  relapsing 


Fio.  173.— Spirillum  Obermeieri  in  blood  of  an  inoculated  ape.    x  700.    (Koch. 

fever  in  healthy  persons  by  inoculating  them  with  blood  containing 
the  spirillum  of  Obermeier.  The  spirilla  are  found  in  the  blood  dur- 
ing the  febrile  paroxysm,  and  for  a  day  or  two,  at  the  outside,  after 


PATHOGENIC   SPIRILLA.  551 

its  termination  ;  sometimes  they  are  present  in  great  numbers,  and 
at  others  can  only  be  found  by  searching  several  microscopic  fields; 
they  are  not  present  in  the  various  secretions — urine,  sweat,  saliva, 
etc.  In  fatal  cases  the  principal  pathological  changes  are  found  in 
the  spleen,  which  is  greatly  enlarged,  and  in  the  liver  and  marrow 
of  the  bones,  which  contain  inflammatory  and  necrotic  foci.  Koch 
and  Carter  have  succeeded  in  transmitting  the  disease  to  monkeys 
by  subcutaneous  inoculations  with  small  amounts  of  defibrinated 
blood  containing  the  spirillum.  After  an  incubation  period  of  seve- 
ral days  typical  febrile  paroxysms  were  developed,  during  which 
the  actively  motile  spirilla  were  found  in  the  blood  in  large  numbers. 
Blood  from  one  animal,  taken  during  the  attack,  induced  a  similar 
febrile  paroxysm  when  inoculated  into  another  of  the  same  species — 
relapses,  such  as  characterize  the  disease  in  man,  were  not  observed. 
One  attack  did  not  preserve  the  animals  experimented  upon  from  a 
similar  attack  when  they  were  again  inoculated  after  an  interval  of 
a  few  days.  Soudakewitch  (1891)  has  made  successful  inoculation 
experiments  in  monkeys,  and  has  shown  that  in  monkeys  from  which 
the  spleen  has  previously  been  removed  the  spirilla  continue  to 
multiply  very  abundantly  in  the  blood  and  the  disease  has  a  fatal 
termination,  whereas  in  monkeys  from  which  the  spleen  has  not  been 
removed  the  spirilla  disappear  from  the  blood  within  a  few  days 
after  the  access  of  the  febrile  paroxysm  and  the  animal  recovers. 

192.   SPIRILLUM  ANSERUM. 

Synonym. — Spirochaeta  anserina  (Sakharoff). 

Obtained  by  Sakharoff  (1890)  from  the  blood  of  geese  affected  by  a  fatal 
form  of  septicaemia  due  to  this  spirillum.  This  disease  prevails  among  geese 
in  Caucasia,  especially  in  swampy  regions,  appearing  annually  and  destroy- 
ing a  large  number  of  the  domestic  geese. 

Morphology. — Resembles  the  spirillum  of  relapsing  fever.  The  long  and 
flexible  spiral  filaments,  when  the  disease  is  at  its  height,  are  often  seen  in 
interlaced  masses,  around  the  margins  of  which  radiate  single  filaments 
which  by  their  movements  cause  the  whole  mass  to  change  its  place,  as  if  it 
were  a  single  organism.  These  masses  are  sometimes  so  large  that  a  single 
one  occupies  the  entire  field  of  the  microscope. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters.  —  An  aerobic,  motile  spirillum.  Not  cultivated 
in  artificial  media.  The  movements  are  very  active,  resembling  those  of 
Spirillum  Obermeieri,  but  cease  in  an  hour  or  two  in  preparations  made  from 
the  blood  of  geese  containing  it. 

Pathogenesis.  —A  small  quantity  of  blood  from  an  infected  goose  inocu- 
lated into  a  healthy  animal  of  the  same  species  induces  the  disease  after  a 
period  of  incubation  of  four  to  five  days.  The  infected  goose  ceases  to  eat, 
becomes  apathetic,  remaining  in  one  place,  and  usually  dies  at  the  end  of  a 
week  ;  the  temperature  is  increased,  and  in  some  cases  there  is  diarrhoea. 
The  spirilla  are  found  in  the  blood  at  the  outset  of  the  malady,  but  after 
death  they  are  not  seen  either  in  the  blood  or  in  the  various  organs.  The 
heart  and  the  liver  are  found  to  have  undergone  a  fatty  degeneration,  and 
yellowish,  cheesy  granules  the  size  of  a  millet  seed  are  seen  upon  the  surface 
of  these  organs.  The  spleen  is  soft  and  easily  broken  up  by  the  fingers. 


552  PATHOGENIC    SPIRILLA. 

Inoculations  into  chickens  and  pigeons  were  without  result  ;  in  one 
chicken  the  spirilla  were  found  in  the  blood  on  the  fourth  day  after  inocula- 
tion, but  the  fowl  recovered. 

193.    SPIRILLUM  CHOLERA  ASIATICS. 

Synonyms. — Spirillum  ("  bacillus  ")  of  cholera  ;  Comma  bacillus 
of  Koch ;  Kommabacillus  der  Cholera  Asiaticae ;  Bacille-virgule 
cholerigene. 

Discovered  by  Koch  (1884)  in  the  excreta  of  cholera  patients  and 
in  the  contents  of  the  intestine  of  recent  cadavers. 

The  researches  of  Koch,  made  in  Egypt  and  in  India  (1884),  and 
subsequent  researches  by  bacteriologists  in  various  parts  of  the 
world,  show  that  this  spirillum — so-called  "  comma  bacillus" — is  con- 
stantly present  in  the  contents  of  the  intestine  of  cholera  patients 
during  the  height  of  the  disease,  and  that  it  is  not  found  in  the  con- 
tents of  the  intestine  of  healthy  persons  or  of  those  suffering  from 


*  J; 


FIG.  174.  FIG.  176. 

FIG.  174.— Spirillum  choleras  Asiaticse.    X  1,000.    From  a  photomicrograph.    (Koch.) 
FIG.  175.— Spirillum  cholera  Asiatic®,  involution  forms.    X  700.    (Van  Ermengem.) 

other  diseases  than  cholera.  The  etiological  relation  of  this  spiril- 
lum to  Asiatic  cholera  is  now  generally  admitted  by  bacteriologists. 
Morphology. — Slightly  curved  rods  with  rounded  ends,  from  0.8 
to  2  /tin  length  and  about  0.3  to  0.4 /tin  breadth.  The  rods  are 
usually  but  slightly  curved,  like  a  comma,  but  are  occasionally  in 
the  form  of  a  half -circle,  or  two  united  rods  curved  in  opposite 
directions  may  form  an  S-shaped  figure.  Under  certain  circum- 
stances the  curved  rods  grow  out  into  long,  spiral  filaments,  which 
may  consist  of  numerous  spiral  turns,  and  in  hanging-drop  cultures 
the  S-shaped  figures  may  also  be  seen  to  form  the  commencement 
of  a  spiral ;  in  stained  preparations  the  spiral  character  of  the  long 
filaments  is  often  obliterated,  or  nearly  so.  When  development  is 
very  rapid  the  short,  curved  rods  or  S-shaped  spirals  only  are  seen  ; 
but  in  hanging-drop  cultures,  or  in  media  in  which  the  develop. 


PATHOGENIC    SPIRILLA. 


55S 


ment  is  retarded  by  an  unfavorable  temperature,  the  presence  of  a 
little  alcohol,  etc.,  the  long,  spiral  filaments  are  quite  numerous,  and 
bacteriologists  generally  agree  that  the  so-called  "  comma  bacillus  n 
is  really  only  a  fragment  of  a  true  spirillum.  By  Loffler's  method 
of  staining  the  rods  may  be  seen  to  have  a  single  terminal  flagel- 
lum.  In  old  cultures  the  bacilli  frequently  lose  their  characteristic 
form  and  become  variously  swollen  and  distorted — involution  forms. 
Hueppe  has  described  the  appearance  of  spherical  bodies  in  the 
course  of  the  spiral  filaments,  which  he  believes  to  be  reproductive 
elements — so-called  arthrospores. 

Stains  with  the  aniline  colors  usually  employed,  but  not  as  quick- 
ly as  many  other  bacteria ;   an  aqueous  solution  of  fuchsin  is  the 


FIG.  176.  FIG.  177. 

FIG.  176.— Spirillum  cholerse  Asiatic®;  colonies  upon  gelatin  plate,  end  of  thirty  hours.  X  100. 
Photograph  by  Frankel  and  Pfeiffer. 

FIG.  177.— Spirillum  cholera  Asiatic®,  from  a  gelatin  culture,  x  1,000.  From  a  photomicro- 
graph. (Frankel  and  Pfeiffer.) 

most  reliable  staining  agent;  is  decolorized  by  iodine  solution — 
Gram's  method.  Sections  may  be  stained  with  Loffler's  solution. 

Biological  Characters. — An  aerobic  (facultative  anaerobic), 
liquefying,  motile  spirillum.  Grows  in  the  usual  culture  media  at 
the  room  temperature — more  rapidly  in  the  incubating  oven.  Does 
not  grow  at  a  temperature  above  42°  or  below  14°  C.  Does  not  form 
endogenous  spores  (forms  arthrospores,  according  to  Hueppe  ?). 

In  gelatin  plate  cultures,  at  22°  C.,  at  the  end  of  twenty-four 
hours  small,  white  colonies  may  be  perceived  in  the  depths  of  the 
gelatin ;  these  grow  towards  the  surface  and  cause  liquefaction  of 
the  gelatin  in  the  form  of  a  funnel  which  gradually  increases  in 
39 


554 


PATHOGENIC   SPIRILLA. 


depth,  and  at  the  bottom  of  which  is  seen  the  colony  in  the  form  of 
a  small,  white  mass  ;  as  a  result  of  this  the  plates  on  the  second  or 
third  day  appear  to  be  perforated  with  numerous  small  holes ;  later 

the  gelatin  is  entirely  liquefied.  Under 
a  low  power  the  young  colonies,  before 
liquefaction  has  commenced,  present  a 
rather  characteristic  appearance ;  they 
are  of  a  white  or  pale-yellow  color,  and 
have  a  more  or  less  irregular  outline, 
the  margins  being  rough  and  uneven; 
the  texture  is  coarsely  granular,  and  the 
surface  looks  as  if  it  were  covered  with 
little  fragments  of  broken  glass,  while 

the  colony  has  a  shining  appearance  ;  when  liquefaction  commences  an 
ill-defined  halo  is  first  seen  to  surround  the  granular  colony,  which 
by  transmitted  light  has  a  peculiar  roseate  hue.  In  stick  cultures  in 
nutrient  gelatin  development  occurs  all  along  the  line  of  inoculation, 


FIG.  178.- Colonies  of  the  cholera 
spirillum;  a,  end  of  twenty  hours;  6, 
end  of  thirty  hours ;  c,  end  of  forty- 
eight  hours;  d,  after  liquefaction  of 
the  gelatin.  (Flugge.) 


Fio.  179.-Spirillum  choleras  Asiaticae;  a,  one  day  old;  6,  three  days  old;  c,  fourdays  old;  d,  five 
days  old ;  e,  seven  days  old ;  /,  10  days  old.    From  photographs  by  Koch. 

but  liquefaction  of  the  gelatin  first  occurs  only  near  the  surface  ;  on 
the  second  day,  at  22°  C.,  a  short  funnel  is  formed  which  has  a 
comparatively  narrow  mouth,  and  the  upper  portion  of  which  con- 
tains air,  while  just  below  this  is  a  whitish,  viscid  mass  ;  later  the 
funnel  increases  in  depth  and  diameter,  and  at  the  end  of  from  four 
to  six  days  may  reach  the  edge  of  the  test  tube ;  in  from  eight  to 
fourteen  days  the  upper  two-thirds  of  the  gelatin  is  completely  lique- 
fied. Owing  to  the  slight  liquefaction  which  occurs  along  the  line  of 
growth  during  the  first  three  or  four  days,  the  central  mass  which 


PATHOGENIC   SPIRILLA. 


555 


had  formed  along  the  line  of  inoculation  settles  down  as  a  curled 
or  irregularly  bent,  yellowish-white  thread  in  the  lower  part  of  a 
slender  tube  filled  with  liquefied  gelatin,  the  upper  part  of  which 
widens  out  and  is  continuous  with  the  funnel  above.  Upon  the  sur- 
face of  nutrient  agar  a  moist,  shining,  white  layer  is  formed  along 
the  line  of  inoculation — impfstrich.  Blood  serum  is  slowly  liquefied 
by  this  spirillum.  Upon  the  surface  of  cooked  potato,  in  the  incu- 
bating oven,  a  rather  thin  and  semi-transparent  brown  or  grayish- 
brown  layer  is  developed.  In  bouillon  the  development  is  rapid  and 
abundant,  especially  in  the  incubating  oven  ;  the  fluid  is  only  slightly 


FIG.  ItiO.— Cultures  in  nutrient  gelatin,  at  the  room  temperature  (16°  to  18°  C.),  at  the  com- 
mencement of  the  fourth  day;  a,  Spirillum  choleree  Asiatic®;  6,  Spirillum  tyrogenum;  c,  Spirillum 
of  Finkler  and  Prior.  (Baumgarten.) 

clouded,  but  the  spirilla  accumulate  at  the  surface,  forming  a  wrin- 
kled membranous  layer.  Sterilized  milk  is  also  a  favorable  culture 
medium.  In  general  this  spirillum  grows  in  any  liquid  containing  a 
small  quantity  of  organic  pabulum  and  having  a  slightly  alkaline 
reaction.  An  acid  reaction  of  the  culture  medium  prevents  its  de- 
velopment, as  a  rule,  but  it  has  the  power  of  gradually  accommo- 
dating itself  to  the  presence  of  vegetable  acids,  and  grows  upon 
potatoes— in  the  incubator  only — which  have  a  slightly  acid  reaction. 
Abundant  development  occurs  in  bouillon  which  has  been  diluted 
with  eight  or  ten  parts  of  water,  and  the  experiments  of  Wolffhugel 


556  PATHOGENIC   SPIRILLA. 

and  Riedel  show  that  it  also  multiplies  to  some  extent  in  sterilized 
river  or  well  water,  and  that  it  preserves  its  vitality  in  such  water 
for  several  months.  But  in  milk  or  water  which  contains  other  bac- 
teria it  dies  out  in  a  few  days.  Gruber  and  Schottelius  have  shown, 
however,  that  in  bouillon  which  is  greatly  diluted  the  cholera  spiril- 
lum may  take  the  precedence  of  the  common  saprophytic  bacteria, 
and  that  they  form  upon  the  surface  of  such  a  medium  the  charac- 
teristic wrinkled  film.  Koch  found  in  his  early  investigations  that  - 
rapid  multiplication  may  occur  upon  the  surface  of  moist  linen,  and  ' 
also  demonstrated  the  presence  of  this  spirillum  in  the  foul  water  of 
a  "  tank "  in  India  which  was  used  by  the  natives  for  drinking 
purposes.  In  the  experiments  of  Bolton  (1886)  the  cholera  spirillum 
was  found  to  multiply  abundantly  in  distilled  water  to  which 
bouillon  was  added  in  the  proportion  of  fifteen  to  twenty-five  parts 
in  one  thousand. 

The  thermal  death-point  of  the  cholera  spirillum  in  recent  cul- 
tures in  flesh-peptone-gelatin,  as  determined  by  the  writer  (1887),  is 
52°  C.,  the  time  of  exposure  being  four  minutes  ;  a  few  colonies  only 
developed  after  exposure  to  a  temperature  of  50°  for  ten  minutes. 
In  Kitasato's  experiments  (1889)  ten  or  even  fifteen  minutes7  expo- 
sure to  a  temperature  of  55°  C.  was  not  always  successful  in  destroy- 
ing the  vitality  of  the  spirillum,  although  in  certain  cultures  exposure 
to  50°  for  fifteen  minutes  was  successful.  He  was  not,  however, 
able  to  find  any  difference  between  old  and  recent  cultures  as  regards 
resistance  to  heat  or  to  desiccation.  In  a  moist  condition  this  spiril- 
lum retains  its  vitality  for  months — as  much  as  nine  months  in  agar 
and  about  two  months  in  liquefied  gelatin.  It  is  quickly  destroyed 
by  desiccation,  as  first  determined  by  Koch,  who  found  that  it  did 
not  grow  after  two  or  three  hours  when  dried  in  a  thin  film  on  a 
glass  cover.  In  Kitasato's  experiments  (1889)  the  duration  of  vital- 
ity was  found  to  vary  from  a  few  hours  to  thirteen  days,  the  differ- 
ence depending  largely  upon  the  thickness  of  the  film.  When  dried 
upon  silk  threads  they  may  retain  their  vitality  for  a  considerably 
longer  time  (Kitasato).  Very  numerous  experiments  have  been 
made  to  determine  the  amount  of  various  disinfecting  agents  re- 
quired to  destroy  the  vitality  of  this  microorganism.  We  give  be- 
low the  results  recently  reported  by  Boer  (1890),  whose  experiments 
were  made  in  Koch's  laboratory.  Experiments  upon  a  culture  in 
bouillon  kept  for  twenty-four  hours  in  the  incubating  oven,  time  of 
exposure  two  hours  :  hydrochloric  acid,  1  : 1,350 ;  sulphuric  acid, 
1  : 1,300 ;  caustic  soda,  1 : 150  ;  ammonia,  1 :  350  ;  mercuric  cyanide, 
1  : 60,000  ;  gold  and  sodium  chloride,  1  : 1,000  ;  silver  nitrate,  1: 4,000; 
arsenite  of  soda,  1  : 400 ;  malachite  green,  1  :  5,000  ;  methyl  violet, 
1  : 1,000  ;  carbolic  acid,  1  : 400  ;  creolin,  1  : 3,000  ;  lysol,  1  : 500.  In 


PATHOGENIC    SPIRILLA.  557 

Bolton's  experiments  (1887)  mercuric  chloride  was  effective  in  two 
hours  in  the  proportion  of  1  : 10,000  ;  sulphate  of  copper,  1  : 500. 

The  low  thermal  death-point  and  comparatively  slight  resisting 
power  for  desiccation  and  chemical  agents  indicate  that  this  spiril- 
lum does  not  form  spores,  and  most  bacteriologists  agree  that  this 
is  the  case.  Hueppe,  however,  has  described  a  mode  of  spore  for- 
mation which  is  different  from  that  which  occurs  among  the  bacilli, 
viz. ,  the  formation  of  so-called  arthrospores  ;  these  are  said  to  be 
developed  in  the  course  of  the  spiral  threads,  not  as  endogenous  re- 
fractive spores,  but  as  spherical  bodies  which  have  a  somewhat 
greater  diameter  than  the  filament  and  are  somewhat  more  refrac- 
tive. This  mode  of  spore  formation  has  not  been  observed  by  Kita- 
sato  and  other  bacteriologists  who  have  given  attention  to  the  Ques- 
tion, and  cannot  be  considered  as  established.  In  competition  with 
the  ordinary  putrefactive  bacteria  the  cholera  spirillum  soon  disap- 
pears, and,  as  determined  by  Neffelrnan  and  by  Kitasato,  they  only 
survive  for  a  few  days  when  mixed  with  normal  faeces. 

A  test  for  the  presence  of  the  cholera  spirillum  has  been  found 
by  Bujwid  and  by  Dunham  in  the  reddish- violet  color  produced  in 
bouillon  cultures  containing  peptone,  or  in  cultures  in  nutrient  gela- 
tin, when  a  small  quantity  of  sulphuric  acid  is  added  to  the  culture. 
According  to  Frankel,  this  test  serves  to  distinguish  it  from  the  ordi- 
nary bacteria  of  the  intestine  and  from  the  Finkler-Prior  spirillum, 
but  not  from  MetschnikofFs  spirillum  ("  vibrio").  The  reaction  is 
shown  by  bouillon  cultures  which  have  been  in  the  incubating  oven 
for  ten  or  twelve  hours,  and  by  gelatin  cultures  in  which  liquefac- 
tion has  occurred.  The  sulphuric  acid  used  should  be  quite  pure  ; 
the  color  quickly  appears  and  is  reddish- violet  or  purplish-red.  Ac- 
cording to  Salkowski,  the  red  color  is  due  to  the  well-known  indol 
reaction,  which  in  cultures  of  the  cholera  spirillum  is  exceptionally 
intense  and  rapid  in  its  development.  A  test  which  is  said  to  dis- 
tinguish cultures  of  the  cholera  spirillum  from  the  spirillum  of  De- 
neke  and  that  of  Finkler-Prior,  has  been  proposed  by  Cahen.  This 
consists  in  adding  a  solution  of  litmus  to  the  bouillon  and  in  making 
the  culture  at  37°  C.  The  cholera  cultures  show  on  the  following 
day  a  decoloration  which  does  not  occur  at  this  temperature  with  the 
other  spirilla  named. 

For  determining  as  promptly  as  possible  whether  certain  suspected 
excreta  contain  cholera  spirilla,  a  little  of  the  material  maybe  used 
to  inoculate  greatly  diluted  bouillon,  gelatin  plates  being  made  at 
the  same  time.  At  the  end  of  ten  or  twelve  hours  the  cholera  spiril- 
lum, if  present,  will  already  have  formed  a  characteristic  wrinkled 
film  upon  the  surface  ;  a  little  of  this  should  be  used  to  start  a  new 
culture  in  diluted  bouillon,  and  a  series  of  gelatin  plates  made  from 


558  PATHOGENIC   SPIRILLA. 

it,  after  which  the  color  test  may  be  applied.  The  result  of  this,  in 
connection  with  the  morphology  of  the  microorganisms  forming  the 
film  and  the  character  of  growth  in  the  gelatin  plates,  will  estab- 
lish the  diagnosis  if  the  cholera  spirillum  is  present  in  considerable 
numbers.  If  but  few  are  present  in  the  original  material  it  may  be 
necessary  to  make  two  or  more  series  of  plates  and  bouillon  cultures 
before  a  pure  culture  can  be  obtained  and  a  positive  diagnosis  made. 

Brieger  has  succeeded  in  isolating  several  toxic  ptomaines  from 
cultures  of  the  cholera  bacillus,  some  of  which  had  previously  been 
obtained  from  other  sources — cadaverin,  putrescin,  creatinin,  me- 
thyl-guanidin.  In  addition  to  these  he  obtained  two  toxic  sub- 
stances not  previously  known.  One  of  these  is  a  diamin,  resembling 
trimethylenediamin  ;  it  gave  rise  to  cramps  and  muscular  tremor  in 
inoculated  animals.  The  other  poison  reduced  the  frequency  of  the 
heart's  action  and  the  temperature  of  the  body  in  the  animals  sub- 
jected to  experiment.  In  more  recent  researches  made  by  Brieger 
and  Frankel  (1890)  a  toxalbumin  was  obtained  from  cholera  cultures 
which,  when  injected  subcutaneously  into  guinea-pigs,  caused  their 
death  in  two  or  three  days,  but  had  no  effect  upon  rabbits. 

Pfeiffer  in  1892  published  his  extended  researches  relating  to  the 
cholera  poison.  He  finds  that  recent  aerobic  cultures  of  the  cholera 
spirillum  contain  a  specific  toxic  substance  which  is  fatal  to  guinea- 
pigs  in  extremely  small  doses.  This  substance  stands  in  close  rela- 
tion with  the  bacterial  cells  and  is  perhaps  an  integral  part  of  the 
same.  The  spirilla  may  be  killed  by  chloroform,  thymol,  or  by  desi- 
cation  without  apparent  injury  to  the  toxic  potency  of  this  sub- 
stance. It  is  destroyed,  however,  by  absolute  alcohol,  by  concen- 
trated solutions  of  neutral  salts,  and  by  the  boiling  temperature,  and 
secondary  toxic  products  are  formed  which  have  a  similar  physio- 
logical action  but  are  from  ten  to  twenty  times  less  potent.  Similar 
toxic  substances  were  obtained  by  Pfeiffer  from  cultures  of  Finkler- 
Prior's  spirillum  and  from  Spirillum  Metschnikovi. 

The  spirillum  is  not  found  in  the  blood  or  in  the  various  organs  of 
individuals  who  have  succumbed  to  an  attack  of  cholera,  but  it  is 
constantly  found  in  the  alvine  discharges  during  life  and  in  the  con- 
tents of  the  intestine  examined  immediately  after  death ;  frequently  in 
almost  a  pure  culture  in  the  colorless  "  rice-water"  discharges.  It  is 
evident,  therefore,  that  if  we  accept  it  as  the  etiological  agent  in  this 
disease,  the  morbid  phenomena  must  be  ascribed  to  the  absorption  of 
toxic  substances  formed  during  its  multiplication  in  the  intestine.  In 
cases  which  terminated  fatally  after  a  very  brief  sickness  Koch  found 
but  slight  changes  in  the  mucous  membrane  of  the  intestine,  which 
was  slightly  swollen  and  reddened ;  but  in  more  protracted  cases  the 
follicles  and  Peyer's  patches  were  reddened  around  their  margins,  and 


PATHOGENIC   SPIRILLA. 


§59 


an  invasion  of  the  mucous  membrane  by  the  "  comma  bacilli  "  was 
observed  in  properly  stained  sections ;  they  penetrated  especially 
the  follicles  of  Lieberkiihn,  and  in  some  cases  were  seen  between  the 
epithelium  and  basement  membrane.  As  a  rule,  the  spirillum  is  not 
present  in  vomited  matters,  but  Koch  found  it  in  small  numbers  in 
two  cases  and  Nicati  and  Rietsch  in  three.  In  about  one  hundred 
cases  in  which  Koch  examined  the  excreta,  or  the  contents  of  the  in- 
testine of  recent  cadavers,  during  his  stay  in  Egypt,  in  India,  and  in 
Toulon,  his  "  comma  bacillus"  was  constantly  found,  and  other  ob- 
servers have  fully  confirmed  him  in  this  particular — Mcati  and 
Rietsch  in  thirty-one  cases  examined  at  Marseilles  ;  Pf  eiffer,  twelve 
cases  in  Paris ;  Schottelius  in  cases  examined  in  Turin ;  Ceci  in 


.-•-'"  ^^^wr 

-  ;£*-- ..  v^7" 

FIG.  181. — Section  through  mucous  membrane  of  intestine  from  cholera  cadaver;  a  tubular 
gland  (a)  is  cut  obliquely;  in  the  interior  of  this  (6),  and  between  the  epithelial  and  basement 
membrane,  are  numerous  spirilla.  X  600.  (Tlugge.) 

Genoa,  etc.  On  the  other  hand,  very  numerous  control  experiments 
made  by  Koch  and  others  show  that  it  is  not  present  in  the  alvine 
discharges  of  healthy  persons  or  in  the  contents  of  the  intestine  of 
those  who  die  from  other  diseases.  In  the  writer's  extended  bacte- 
riological studies  of  the  excreta,  and  contents  of  the  intestine  of  ca- 
davers, in  yellow  fever,  he  has  not  once  encountered  any  microor- 
ganism resembling  the  cholera  spirillum. 

As  none  of  the  lower  animals  are  liable  to  contract  cholera  during 
the  prevalence  of  an  epidemic,  or  as  a  result  of  the  ingestion  of  food 
contaminated  with  choleraic  excreta,  we  have  no  reason  to  expect 
that  pure  cultures  of  the  spirillum  introduced  by  subcutaneous  inocu- 
lation or  by  the  mouth  will  give  rise  in  them  to  a  typical  attack  of 


560  PATHOGENIC   SPIRILLA. 

cholera.     Moreover,  it  has  been  shown  by  experiment  that  this  spi- 
rillum is  very  sensitive  to  the  action  of  acids,  and  is  quickly  de- 
stroyed by  the  acid  secretions  of  the  stomach,  of  man  or  the  lower 
animals,  when  the  functions  of  this  organ  are  normally  performed. 
By  a  special  method  of  procedure,  however,  Nicati  and  Bietsch,  and 
Koch,  have  succeeded  in  producing  in  guinea-pigs  choleraic  symp- 
toms and  death.     The  first-named  investigators  injected  cultures  of 
the  spirillum  into  the  duodenum,  after  first  ligating  the  biliary  duct; 
the  animals  experimented  upon  died,  and  the  intestinal  contents  con- 
tained the  spirillum  in  large  numbers.     The  fact  that  this  procedure 
involves  a  serious  operation  which  alone  might  be  fatal,  detracts 
from  the  value  of  the  results  obtained.     Koch's  experiments  on 
guinea-pigs  are  more  satisfactory,  and,  having  been  fully  controlled 
by  comparative  experiments,  show  that  the  "  comma  bacillus "  is 
pathogenic  for  these  animals  when  introduced  in  a  living  condition 
into  the  intestine.     This  was  accomplished  by  first  neutralizing  the 
contents  of  the  stomach  with  a  solution  of  carbonate  of  soda — five 
cubic  centimetres  of  a  five-per-cent  solution,  injected  into  the  stomach 
through  a  pharyngeal  catheter.     For  the  purpose  of  restraining  in- 
testinal peristalsis  the  animal  also  receives,  in  the  cavity  of  the  abdo- 
men, a  tolerably  large  dose  of  laudanum — one  gramme  tincture  of 
opium  to  two  hundred  grammes  of  body  weight.     The  animals  are 
completely  narcotized  by  this  dose  for  about  half  an  hour,  but  re- 
cover from  it  without  showing  any  ill  effects.     Soon  after  the  ad- 
ministration of  the  opium  a  bouillon  culture  of  the  cholera  spirillum 
is  injected  into  the  stomach  through  a  pharyngeal  catheter.     As  a 
result  of  this  procedure  the  animal  shows  an  indisposition  to  eat  and 
other  signs  of  sickness,  its  posterior  extremities  become  weak  and 
apparently  paralyzed,  and,  as  a  rule,  death  occurs  within  forty-eight 
hours.     At  the  autopsy  the  small  intestine  is  found  to  be  congested 
and  is  filled  with  a  watery  fluid  containing  the  spirillum  in  great 
numbers.     Comparatively  large  quantities  of  a  pure  culture  injected 
into  the  abdominal  cavity  of  rabbits  or  of  mice  often  produce  a  fatal 
result  within  two  or  three  hours  ;   and  Nicati  and  Bietsch  have  ob- 
tained experimental  evidence  of  the  pathogenic  power  of  filtered  cul- 
tures not  less  than  eight  days  old.     The  most  satisfactory  evidence 
that  this  spirillum  is  able  to  produce  cholera  in  man  is  afforded  by  an 
accidental  infection  which  occurred  in  Berlin  (1884),  in  the  case  of  a 
young  man  who  was  one  of  the  attendants  at  the  Imperial  Board  of 
Health  when  cholera  cultures  were  being  made  for  the  instruction  of 
students.     Through  some  neglect  the  spirillum  appears  to  have  been 
introduced  into  his  intestine,  for  he  suffered  a  typical  attack  of 
cholera,  attended  by  thirst,  frequent  watery  discharges,  cramps  in 
the  extremities,  and  partial  suppression  of  urine.     Fortunately  he 


PATHOGENIC   SPIRILLA.  561 

recovered  ;  but  the  genuine  nature  of  the  attack  was  shown  by  the 
symptoms  and  by  the  abundant  presence  of  the  "  comma  bacillus" 
in  the  colorless,  watery  discharges  from  his  bowels.  Nicati  and 
Rietsch  observed  a  certain  degree  of  attenuation  in  the  pathogenic 
power  of  the  spirillum  after  it  had  been  cultivated  for  a  considerable 
time  at  20°  to  25°  C.  ;  and  the  observation  has  since  been  made  that 
cultures  which  have  been  kept  up  from  Koch's  original  stock  have 
no  longer  the  primitive  pathogenic  potency. 

Cunningham,  as  a  result  of  researches  made  in  Calcutta  (1891), 
arrives  at  the  conclusion  that  Koch's  "comma  bacillus"  cannot 
be  accepted  as  the  specific  etiological  agent  in  this  disease.  This 
conclusion  is  based  upon  the  results  of  his  own  bacteriological 
studies,  which  may  be  summed  up  as  follows  :  First,  in  many  un- 
doubted cases  of  cholera  he  has  failed  to  find  comma  bacilli.  Sec- 
ond, in  one  case  he  found  three  different  species.  Third,  in  one  case 
the  reaction  with  acids  could  not  be  obtained.  From  sixteen  cases 
in  which  Cunningham  made  cultures  he  obtained  ten  different  vari- 
eties of  comma  bacilli,  the  characters  of  which  he  gives  in  his  pub- 
lished report.  It  may  be  that  in  India,  which  appears  to  be  the 
permanent  habitat  of  the  cholera  spirillum,  many  varieties  of  this 
microorganism  exist ;  but  extended  researches  made  in  the  laborato- 
ries of  Europe  show  that  Cunningham  is  mistaken  in  supposing  that 
spirilla  resembling  Koch's  "  comma  bacillus  "  are  commonly  present 
in  the  intestine  of  healthy  persons.  The  view  advocated  is  that 
during  the  attack  these  spirilla  are  found  in  increased  numbers  be- 
cause conditions  are  more  favorable  for  their  development,  but  that 
they  have  no  etiological  import.  The  writer  would  remark  that,  in 
very  extended  researches  made  in  the  United  States  and  in  Cuba,  he 
has  never  found  any  microorganism  resembling  Koch's  cholera  spi- 
rillum in  the  faeces  of  patients  with  yellow  fever  or  of  healthy  indi- 
viduals, or  in  the  intestinal  contents  of  yellow-fever  cadavers. 

194:.    SPIRILLUM   OF   FINKLER  AND   PRIOR. 

Synonym. — Vibrio  proteus. 

Obtained  by  Finkler  and  Prior  (1884)  from  the  faeces  of  patients  with 
cholera  noslras,  after  allowing  the  dejecta  to  stand  for  some  days.  Subse- 
quent researches  have  not  sustained  the  view  that  this  spirillum  is  the  speci- 
fic cause  of  cholera  morbus. 

Morphology.— Resembles  the  spirillum  of  Asiatic  cholera,  but  the  curved 
segments  ("  bacilli"  )  are  somewhat  longer  and  thicker  and  not  so  uniform 
in  diameter,  the  central  portion  being  usually  thicker  than  the  somewhat 
pointed  ends ;  forms  spiral  filaments,  which  are  not  as  numerous,  and  are 
usually  shorter  than  those  formed  by  the  cholera  spirillum.  In  unfavorable 
media  involution  forms  are  common — large  oval,  spherical,  or  spindle 
shaped  cells,  etc.  Has  a  single  flagellum  at  one  end  of  the  curved  segments, 
which  is  from  one  to  one  and  one-half  times  as  long  as  these. 

Stains  with  the  usual  aniline  colors — best  with  an  aqueous  solution  of 
fuchsin. 


562 


PATHOGENIC   SPIRILLA. 


Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  spirillum.  Spore  formation  not  demonstrated.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  small, 
white,  punctiform  colonies  are  developed  at  the  end  of  twenty  four  hours, 

n  to  be  finely  granular  and  yellowish  or 


which  under  the  microscope  are  seen 

yellowish-brown  in  color ;  liquefaction  of  the  gelatin  around  these  colonies 
progresses  rapidly,  and  at  the  end  of  forty-eight  hours  is  usually  complete  in 
plates  where  they  are  numerous.  Isolated  colonies  on  the  second  day  form 
saucer-shaped  depressions  in  the  gelatin  the  size  of  lentils,  having  a  sharply 
defined  border.  In  gelatin  stick  cultures  liquefaction  progresses  much  more 
rapidly  than  in  similar  cultures  of  the  cholera  spirillum,  and  a  stocking- 
shaped  pouch  of  liquefied  gelatin  is  already  seen  on  the  second  day,  which 
rapidly  increases  in  dimensions,  so  that  by  the  end  of  a  week  the  gelatin  is 
usually  completely  liquefied ;  upon  the  surface  of  the  liquefied  medium  a 
whitisn  film  is  seen.  Upon  agar  a  moist,  slimy  layer,  covering  the  entire 
surface,  is  quickly  developed.  The  growth  in  blood  serum  is  rapid  and 


d 


FIG.  182. 


FIG.  183. 


FIG.  184. 


FIG.  188.— Spirillum  of  Finkler  and  Prior,  from  a  gelatin  culture.  X  1,000.  From  a  photomicro- 
graph. CFriinkel  and  Pfeiffer.) 

FIG.  188.— Spirillum  of  Finkler  and  Prior;  colonies  upon  gelatin  plate;  a,  end  of  sixteen  hours; 
5,  end  of  twenty-four  hours;  c,  end  of  thirty-six  hours.  X  80.  (FlQ«ge  ) 

FIG.  184.— Spirillum  of  Finkler  and  Prior;  culture  in  nutrient  gelatin;  c,  two  days  old;  d,  four 
days  old.  (Flugge.) 

causes  liquefaction  of  the  medium.  Upon  potato  this  spirillum  grows  at  the 
room  temperature  and  produces  a  slimy,  gravish-yellow,  glistening  layer, 
which  soon  extends  over  the  entire  surface.  The  cholera  spirillum  does  not 
grow  upon  potato  at  the  room  temperature.  The  cultures  of  the  Fink  In- 
Prior  spirillum  give  off  a  tolerably  strong  putrefactive  odor,  and,  according 
to  Buchner,  in  media  containing  sugar  an  acid  reaction  is  produced  as  a  re- 
sult of  their  development.  They  have  a  greater  resistance  to  desiccation  tl  uin 
the  cholera  spirillum. 

Pathogenesis. — Pathogenic  for  guinea-pigs  when  injected  into  the 
stomach  by  Koch's  method,  after  previous  injection  of  a  solution  of  car- 
bonate of  soda,  but  a  smaller  proportion  of  the  animals  die  from  sucli  injec- 
tions (Koch).  At  the  autopsy  the  intestine  is  pale,  and  its  watery  contents, 


PATHOGENIC   SPIRILLA.  563 

which  contain  the  spirilla  in  great  numbers,  have  a  penetrating-,  putrefactive 
odor. 

195.    SPIRILLUM   TYROGENUM. 

Synonyms — Spirillum  of  Deneke;  Kasespirillen. 
Obtained  by  Deneke  (1885)  from  old  cheese. 

Morphology. — Curved  rods  and  long,  spiral  filaments  resembling  the 
spirilla  of  Asiatic  cholera.    The  diameter  of  the  curved  segments  is  some- 
what less  than  that  of  the  cholera  spirillum,  and  the  turns  in  the  spiral  fila- 
ments are  lower  and  closer  together.    The  diame- 
ter of  the  "commas"  is  uniform  throughout,  so 
that  this  spirillum  more  closely  resembles  the          ~ 
cholera  spirillum  than  does  that  of  Finkler  and 
Prior. 

Stains  with  the  usual  aniline  colors— best 
with  an  aqueous  solution  of  f uchsin. 

Biological  Characters. — An  aerobic  and  fac- 
ultative anaerobic,  liquefying,  motile  spirillum. 
Spore  formation  not  demonstrated.  Grows  in 

the  usual  culture  media  at  the  room  temperature        FIG.  185.  —  Spirillum  tyroge- 
— more  rapidly  than  the  cholera  spirillum  and     num.    x  700.   (Flugge.) 
less  so  than  that  of  Finkler  and  Prior.      Upon 

gelatin  plates  small,  punctiform  colonies  are  developed,  which  on  the  second 
day  are  about  the  size  of  a  pin's  head  and  have  a  yellowish  color ;  under 
the  microscope  they  are  seen  to  be  coarsely  granular,  of  a  yellowish -green 
color  in  the  centre  and  paler  towards  the  margins.  The  outlines  of  the  colo- 
nies are  sharply  defined  at  first,  but  later,  when 
liquefaction  has  commenced,  the  sharp  contour 
is  no  longer  seen.  At  first  liquefaction  of  the 
gelatin  causes  funnel-shaped  cavities  resembling 
those  formed  by  the  cholera  spirillum,  but  lique- 
faction is  more  rapid.  In  gelatin  stick  cultures 
.  liquefaction  occurs  all  along  the  line  of  punc- 

ture, and  the  spirilla  sink  to  the  bottom  of  the 

FIG  i86.-SPiriliumtyrogenum;  liquefied  gelatin  in  the  form  of  a  coiled  mass, 
colonies  in  gelatin  plate;  a,  end  while  a  thin,  yellowish  layer  forms  upon  the 
of  sixteen  hours;  6,  end  of  twen-  surface ;  complete  liquefaction  usually  occurs  in 

ty-four  hours;  c,  end  of  thirty-       about  two  weeks,      Upon  the  surface  of  agar  a 

thin,   yellowish  layer   forms  along  the    impf- 
strich.     Upon  potato,  at  a  temperature  of  37°  C., 

a  thin,  yellow  layer  is  usually  developed   (not    always— Eisenberg) ;   this 

contains,  as  a  rule,  beautifully  formed,  long,  spiral  filaments. 

Pathogenesis. — Pathogenic    for    guinea-pigs  when  introduced  into  the 

stomach  by  Koch's  method;  three  out  of  fifteen  animals  treated  in  this  way 

succumbed. 

196.    SPIRILLUM  METSCHNIKOVI. 

Synonym. — Vibrio  Metschnikovi  (Gameleia). 

Obtained  by  Gameleia  (1888)  from  the  intestinal  contents  of  chickens 
dying  of  an  infectious  disease  which  prevails  in  certain  parts  of  Russia  dur- 
ing the  summer  months,  and  which  in  some  respects  resembles  fowl  cholera. 
The  experiments  of  Gameleia  show  that  the  spirillum  under  consideration  is 
the  cause  of  the  disease  referred  to,  which  he  calls  gastro-enteritis  cholerica. 

Morphology. — Curved  rods  with  rounded  ends,  and  spiral  filaments;  the 
curved  segments  are  usually  somewhat  shorter,  thicker,  and  more  decidedly 
curved  than  the  "  comma  bacillus  "  of  Koch.  The  size  differs  very  consid- 
erably in  the  blood  of  inoculated  pigeons,  the  diameter  being  sometimes 
twice  as  great  as  that  of  the  cholera  spirillum,  and  at  others  about  the  same. 
A  single,  long,  undulating  flagellum  maybe  seen  at  one  extremity  of  the 
spiral  filaments  or  curved  rods  in  properly  stained  preparations. 


564 


PATHOGENIC   SPIRILLA. 


Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 
Biological  Characters. — An  aerobic  (facultative  anaerobic  ?),  liquefy- 
ing, motile  spirillum.  According  to  Gamaleia,  endogenous  spores  are  formed 
by  this  spirillum;  but  Pfeiffer  does  not  confirm  this  observation,  and  it  must 
,  be  considered  extremely  doubtful  in  view  of  the  slight 
resistance  to  heat— killed  in  five  minutes  by  a  temperature 
of  50°  C.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  Upon  gelatin  plates  small,  white,  puncti- 
form  colonies  are  developed  at  the  end  of  twelve  to  six- 
teen hours  ;  these  rapidly  increase  in  size  and  cause  lique- 
faction of  the  gelatin,  which  is,  however,  much  more  rapid 
with  some  than  with  others.  At  the  end  of  three  days 
large,  saucer-like  areas  of  liquefaction  may  be  seen,  resem- 
bling that  produced  by  the  Finkler-Prior  spirillum  and  the 
contents  of  which  are  turbid,  while  other  colonies  have 
produced  small,  funnel-shaped  cavities  filled  with  trans- 
parent, liquefied  gelatin  and  resembling  colonies  of  the 
cholera  spirillum  of  the  same  age.  Under  the  microscope 
the  larger  liquefied  areas  are  seen  to  contain  yellow ish- 
brown  granular  masses  which  are  in  active  movement,  and 
the  margins  are  surrounded  by  a  border  of  radiating  fila- 
ments. In  gelatin  stick  cultures  the  growth  resembles  that 
of  the  cholera  spirillum,  but  the  development  is  more  rap- 
id. Upon  agar,  at  37°  C.,  a  yellowish  layer  resembling 
that  formed  by  the  cholera  spirillum  is  quickly  developed. 
Upon  potato  no  growth  occurs  at  the  room  temperature, 
but  at  37°  C.  a  yellowish-brown  or  chocolate-colored  layer 
is  formed,  which  closely  resembles  that  produced  by  the 
cholera  spirillum  under  the  same  circumstances.  In  bouil- 
lon, at  37°  C.,  development  is  extremely  rapid,  and  the 
liquid  becomes  clouded  and  opaque,  having  a  grayish- white 
color,  while  a  thin,  wrinkled  film  forms  upon  the  surface. 
When  muriatic  or  sulphuric  acid  is  added  to  a  culture  in 
peptonized  bouillon  a  red  color  is  produced  similar  to  that 

froduced  in  cultures  of  the  cholera  spirillum,  and  even  more  pronounced, 
n  milk,  at  35°  C. ,  rapid  development  occurs,  and  the  milk  is  coagulated  at 
the  end  of  a  week ;  the  precipitated  casein  accumulates  at  the  bottom  of  the 
tube  in  irregular  masses  and  is  not  redissplved.    The  milk  acquires  a  strongly 
acid  reaction  and  the  spirilla  quickly  perish. 

Patliogenesis. — Pathogenic  for  chickens,  pigeons,  and  guinea-pigs;  rab- 
bits and  mice  are  refractory  except  for  very  large  doses.  Chickens  suffering 
from  the  infectious  disease  caused  by  this  spirillum  remain  quiet  and  somno- 
lent, with  ruffled  feathers ;  thev  have  diarrhoea ;  the  temperature  is  not  ele- 
vated above  the  normal,  as  is  the  case  in  chicken  cholera.  At  the  autopsy 
the  most  constant  appearance  is  hyperaemia  of  the  entire  alimentary  canal 
A  grayish-yellow  liquid,  more  or  less  mixed  with  blood,  is  found  in  con- 
siderable quantity  in  the  small  intestine ;  the  spleen  is  not  enlarged  and  the 
organs  generally  are  normal  in  appearance.  In  adult  chickens  the  spirillum 
is  not  found  in  the  blood,  but  in  young-  ones  its  presence  may  be  verified  by 
the  culture  method  and  by  inoculation  into  pigeons,  which  die  in  from 
twelve  to  twenty  hours  after  being  inoculated  with  two  to  four  cubic  cen- 
timetres. The  pathological  appearances  in  pigeons  correspond  with  those 
found  in  chickens,  but  usually  the  spirillum  is  found  in  great  numbers  in 
blood  taken  from  the  heart.  A  few  drops  of  a  pure  culture  inoculated  sub 
cutaneously  in  pigeons  or  injected  into  the  muscles  cause  their  death  in 
eight  to  twelve  hours.  Gameleia  claims  that  the  virulence  of  cultures  is 
greatly  increased  by  successive  inoculations  in  pigeons,  but  Pfeiffer  has 
shown  that  very  minute  doses  are  fatal  to  pigeons  and  that  no  decided  in- 
crease of  virulence  occurs  as  a  result  of  successive  inoculations.  According 
to  Gameldia,  chickens  may  be  infected  by  giving  them  food  contaminated 


FIG  187.-Spiri)- 
lum  Metschnikovi; 
culture  in  nutrient 
gelatin, end  of  forty- 
eight  hours  From  a 
photograph.  (Fran- 
kel  and  Pfeiffer.) 


PATHOGENIC   SPIRILLA.  565 


with  the  cultures  of  the  spirillum,  but  pigeons  resist  infection  in  this  way. 
Guinea-pigs  usually  die  in  from  twenty  to  twenty-four  hours  after  receiving 
a  subcutaneous  inoculation  ;  at  the  autopsy  an  extensive  subcutaneous 
oedema  is  found  in  the  vicinity  of  the  point  of  inoculation,  and  a  superficial 
necrosis  may  be  observed  ;  the  blood  and  the  organs  generally  contain  the 
"  vibrio  "  in  great  numbers,  showing  that  the  animals  die  from  general  in- 
fection— acute  septicaemia.  When  infection  occurs  in  these  animals  by  way 
of  the  stomach  the  intestine  will  be  found  highly  inflamed  and  its  liquid  con- 
tents will  contain  numerous  spirilla. 

Gameleia  has  shown  that  pigeons  and  guinea-pigs  may  be  made  immune 
by  inoculating  them  with  sterilized  cultures  of  the  spirillum — sterilized  by 
heat  at  100°  C.  Old  cultures  contain  more  of  the  toxic  substance  than  those 
of  recent  date.  Thus  two  to  three  cubic  centimetres  of  a  culture  twenty  days 
old  will  kill  a  guinea-pig  when  injected  subcutaneously,  while  five  cubic 
centimetres  of  a  culture  five  days  old  usually  fail  to  do  so.  According  to 
Pfeiffer,  old  cultures  have  a  decidedly  alkaline  reaction,  and  their  toxic  power 
is  neutralized  by  the  addition  of  sulphuric  acid. 

Gameleia  has  claimed  that  by  passing  the  cholera  spirillum  of  Koch 
through  a  series  of  pigeons,  by  successive  inoculation,  its  pathogenic  power 
is  greatly  increased,  and  that  when  sterilized  cultures  of  this  virulent  vari- 
ety of  the  ' '  comma  bacillus  "  are  injected  into  pigeons  they  become  immune 
against  the  pathogenic  action  of  the  "  vibrio  Metschnikoff , "  and  the  reverse. 
Pfeiffer  (1889),  in  an  extended  and  carefully  conducted  research,  was  not 
able  to  obtain  any  evidence  in  support  of  this  claim. 

NOTES  RELATING  TO  THE  PATHOGENIC  SPIRILLA. 

During  the  past  three  or  four  years  quite  a  number  of  spirilla 
have  been  obtained  from  various  sources  which  resemble  more  or 
less  closely  the  spirillum  of  Asiatic  cholera.  It  appears  probable 
that  some  of  these  are  in  fact  varieties  of  Koch's  "  comma  bacillus  " 
which  have  undergone  various  modifications  as  a  result  of  the  con- 
ditions under  which  they  have  maintained  their  existence  as  sapro- 
phytes. Others  are  evidently  essentially  different,  and  have  no  very 
near  relationship  to  the  cholera  spirillum.  The  principal  points  of 
difference  between  these  recently  described  spirilla  and  Spirillum 
cholerse  Asiatics  are  given  in  the  following  resume,  for  which  we 
are  indebted  to  Dieudonne  (1894). 

"Since  the  outbreak  of  cholera  in  1892,  various  vibrios  have  been  de- 
scribed which  resemble  more  or  less  closely  the  cholera  vibrio.  When  these 
are  tested  as  to  their  morphological  characters,  growth  in  peptone  solutions, 
in  gelatin  and  agar  plates,  cholera-red  reaction,  and  pathogenic  power,  they 
may  be  divided,  at  the  outset,  into  two  groups  :  viz.,  such  vibrios  as  show 
only  a  remote  resemblance  to  the  cholera  vibrio,  and  therefore  are  easily  dif- 
ferentiated from  it,  and  such  as  present  only  minor  differences  or  none  at 
all  that  have  been  demonstrated. 

* '  To  the  first  group  belongs  the  spirillum  isolated  by  Russell  from  sea 
water — Spirillum  marinum — which  rapidly  liquefies  gelatin  and  does  not 
grow  at  the  body  temperature.  Renon  isolated  from  water,  obtained  at  Bil- 
lancourt,  a  vibrio  which  likewise  quickly  liquefies  gelatin,  but  is  not  patho- 
genic for  guinea-pigs,  either  by  subcutaneous  or  intraperitoneal  inoculation. 
Gimther,  in  examining  the  Spree  water,  found  a  vibrio  which,  upon  gelatin 
plates,  formed  circular  colonies  with  smooth  margins,  very  finely  granular 
and  of  a  brown  color.  This  vibrio  did  not  give  the  indol  reaction,  and  all 
infection  experiments  gave  a  negative  result.  Gunther  named  this  sapro- 


566  PATHOGENIC   SPIRILLA. 

phyte  Vibrio  aquatilis.  About  the  same  time  (1892)  Kiessling  obtained  from 
water,  from  Blankenese,  a  vibrio  which  presented  similar  characters  and 
probably  is  identical  with  that  of  Gunther.  Weibel  obtained  from  well-water 
a  vibrio  which  liquefies  gelatin  more  rapidly  than  the  cholera  vibrio ;  its 
pathogenic  action  was  not  tested.  Bujwid  (1893)  isolated  from  Weichsel 
water  a  vibrio  which  at  low  temperatures  (12°  C.)  grew  almost  the  same  as 
the  cholera  vibrio,  but  at  higher  temperatures  was  easily  distinguished  from 
it.  Bujwid's  assistant,  Orlowski,  found  in  a  well  at  Lubin  a  very  similar 
vibrio.  Loffler  (1893)  obtained  from  the  Peene  water  a  vibrio  which  at  37° 
C.  grows  rapidlv  and  liquefies  gelatin  very  rapidly,  like  the  Finkler-Prior 
spirillum.  Fokker  (1893),  from  water  of  the  harbor  at  Groningen,  obtained 
a  vibrio  which  rapidly  liquefied  gelatin  and  occasionally  gave  the  indol  re- 
action. Injections  into  the  peritoneal  cavity  of  mice  and  guinea-pigs  gave 
a  negative  result.  Fokker  supposes  that  this  is  an  attenuated  cholera  bacil- 
lus, because  it  forms  the  same  ensyme  as  cholera  bacteria,  and  when  culti- 
vated for  three  months  its  characters,  especially  its  peptonizing  power,  had 
changed.  Fischer  (1893)  found  in  the  stools  of  a  woman  suffering  from  diar- 
rhoea a  vibrio  which  in  gelatin  cultures  resembled  that  of  Fiiikler  and 
Prior.  In  bouillon  and  peptone  solution  it  caused  clouding  and  formation  of 
a  pellicle,  but  only  gave  a  slight  indol  reaction.  A  portion  of  the  mice  in- 
oculated subcutaneously  had  after  a  time  abscesses,  from  the  contents  of 
which  Fischer  was  able  to  cultivate  his  vibrio,  which  he  named  Vibrio  helco- 
genes.  Vogler  (1893),  in  an  extended  series  of  examinations  of  faeces,  found 
a  vibrio  which  showed  many  points  of  resemblance  to  the  cholera  vibrio  in 
its  growth  in  gelatin.  But  it  constantly  gave  a  negative  indol  reaction,  and 
was  not  pathogenic  for  guinea-pigs  when  injected  into  the  peritoneal  cavity. 
Bleisch  obtained  from  the  dejecta  of  a  man  who  died  with  choleraic  symptoms 
a  bacterium  which  upon  gelatin  plates  grew  at  first  like  the  cholera  bacillus,  but 
was  distinguished  from  it  by  many  points  of  difference  in  other  respects : 
short  rods,  sometimes  bent,  but  never  showing  spiral  forms.  It  gave  the 
cholera-red  reaction.  Wolf  (1883)  obtained  from  cervical  secretion,  from  a 
woman  suffering  from  chronic  endometritis,  a  comma-formed  bacillus,  which 
in  its  growth  on  gelatin  plates  resembled  the  cholera  vibrio.  The  liquefac- 
tion was,  however,  much  more  rapid,  a  culture  a  day  old  being  as  far  ad- 
vanced as  a  cholera  culture  of  three  to  four  days.  The  addition  of  sulphuric 
acid  to  a  bouillon  culture  caused  a  faint  rose-red  color,  which  upon  standing 
changed  to  brown.  The  addition  of  sulphuric  acid  and  potassium  iodide  paste 
did  not  cause  a  blue  color,  so  there  was  no  formation  of  nitrites.  Bonhoff 
(1893),  in  water  from  Stolpe,  in  Pommerania,  discovered  two  vibrios,  one  of 
which  in  the  first  twenty-four  hours  grew  like  the  cholera  vibrio,  but  did  not 
give  the  cholera-red  reaction.  Out  of  four  guinea-pigs  inoculated  one  only 
died  with  cholera-like  symptoms.  The  other  vibrio  gave  the  cholera-red  reac- 
tion, but  did  not  liquefy  gelatin  and  was  very  inconstant  as  regards  its  patho- 
genic power.  Zorkendorfer  (1893)  isolated  a  vibrio  from  the  stools  of  a 
woman  who  died  with  choleraic  symptoms,  which  ac  first  grew  upon  gelatin 
plates  like  the  cholera  vibrio,  but  after  the  second  day  liquefied  the  gelatin 
very  rapidly,  so  that  it  could  no  longer  be  taken  for  the  same.  The  indol 
reaction  was  constantly  absent,  and  it  was  not  pathogenic  for  guinea-pigs, 
rabbits,  or  pigeons.  Blackstein  (1893)  obtained  from  trie  water  of  the  Seine 
a  comma  bacillus  which  resembled  the  cholera  vibrio  in  many  particulars,  but 
was  distinguished  by  the  finer  granulation  and  more  opaque  appearance  of 
its  colonies.  Sanarelli  (1893),  by  the  use  of  special  media,  isolated  from  the 
water  of  the  Seine  and  of  the  Marne  no  less  man  thirty-two  vibrios,  four  of 
which  resembled  the  cholera  vibrio  in  giving  the  indol  reaction.  Three 
others  gave  the  indol  reaction  after  eight  days  ;  the  remainder  did  not  give  it 
at  all,  or  only  very  faintly.  The  vibrios  which  upon  a  first  inoculation  gave 
no  results  or  only  very  slight  evidence  of  pathogenic  power,  when  carried 
through  a  series  of  animals  caused  a  fatal  infection.  When  a  sterilized  cul- 
ture of  the  colon  bacillus  was  injected  at  the  same  time  death  always  oc- 


PATHOGENIC   SPIRILLA.  567 

curred.  Sanarelli  believes  that  these  vibrios  must  have  had  a  common  ori- 
gin—from the  dejecta  of  cholera  patients.  Fischer  (1894)  has  described  a 
number  of  vibrios  from  sea-water  which  are  distinguished  from  the  cholera 
vibrio  especially  by  a  preference  for  media  containing  sea- water.  Finally, 
the  vibrios  found  in  water,  referred  to  by  Koch  ('  Ueber  den  augenblicklichen 
Stand  der  Cholera-diagnose,'  Zeitschr.  fur  Hygiene,  Bd.  xiv.,  page  319), 
belong  here. 

"Quite  different  from  these  is  a  second  group  of  vibrios  which  in  their  in- 
vestigation offered  great  and  often  almost  insuperable  difficulties  for  the 
differential  diagnosis.  Here,  first  of  all,  is  the  Vibrio  Berolinensis,  found  by 
Neisser  in  August,  1893,  and  described  by  Rubner,  Neisser,  and  Giinther. 
This  was  isolated  from  water  which  had  previously  contained  cholera  vibrios, 
for  which  reason  Dunbar  considers  it  not  impossible  that  this  is  a  genuine 
cholera  vibrio,  somewhat  changed  perhaps  by  long-continued  development 
in  water.  Neither  in  its  morphology  nor  in  its  behavior  in  gelatin  stick  cul- 
tures, in  milk  and  other  media,  could  it  be  distinguished  from  the  genuine 
comma  bacillus  ;  the  indol  reaction  and  pathogenic  action  upon  guinea-pigs 
were  the  same  ;  on  the  contrary,  a  differentiation  was  easily  made  in  gelatin 
plate  cultures.  At  the  end  of  twenty -four  hours  it  formed  small,  spherical, 
finely  granular  colonies,  which  at  the  end  of  forty-eight  hours  were  not  yet 
visible  to  the  naked  eye.  Heider  (1893)  isolated  from  the  water  of  the  Donau 
canal  a  vibrio  which  he  called  Vibrio  Danubicus.  This  resembles  the  chol- 
era vibrio  fully  in  its  morphology.  As  a  distinguishing  character  it  was 
found  that  this  vibrio,  in  thinly  planted  plates,  forms  flat,  superficial  colo- 
nies having  irregularly  rounded  margins  and  other  slight  differences ;  also 
the  pathogenic  action  upon  mice  inoculated  subcutaneously,  and  the  ease  with 
which  guinea-pigs  are  infected  by  way  of  the  respiratory  passages.  It  is 
worthy  of  note  that  the  day  after  the  sample  was  taken  a  man  was  taken  sick 
with  cholera  who  had  worked  on  the  Donau  the  day  before — on  the  principal 
stream  at  a  place  far  below  the  junction  of  the  canal.  Dunbar  (1893)  found 
vibrios  in  the  Elbe,  in  the  Rhine,  in  the  Pegnitz,  and  in  the  Amstel  at  Amster- 
dam. These  presented  no  decided  characters  by  which  he  was  able  to  differ- 
entiate them  from  the  cholera  vibrio.  The  most  careful  comparative  investi- 
gations did  not  lead  to  the  discovery  of  any  points  of  difference  which  had 
not  already  been  observed  in  genuine  cholera  cultures.  Everything,  there- 
fore, indicated  that  these  were  genuine  cholera  bacilli,  especially  as  these 
vibrios  disappeared  from  the  rivers  when  cholera  ceased  to  prevail.  It  was 
first  possible  through  an  observation  of  Kutscher's  to  differentiate  a  portion 
of  these  water  bacteria,  and  certain  vibrios  isolated  from  the  discharges  of 
persons  suspected  of  having  cholera  from  cultures  of  the  cholera  spirillum. 
In  the  presence  of  oxygen,  at  a  suitable  temperature,  they  give  off  a  greenish- 
white  phosphorescence. 

'  'As  phosphorescence  has  never  been  observed  in  undoubted  cholera  cul- 
tures, we  can  assert  with  tolerable  certainty  that  such  phosphorescent  vibrios 
are  not  genuine  cholera  bacteria.  But  as  this  phosphorescent  property  was 
inconstant  in  thirty-eight  out  of  sixty-eight  cultures,  Dunbar  believes  that 
some  reserve  must  be  exercised  in  accepting  this  as  evidence  that  these  are 
not  genuine  cholera  vibrios.  Maassen  (1894)  gives  as  a  further  distinguishing 
character  of  these  phosphorescent  vibrios  the  fact  that  they  form  a  strong, 
usually  wrinkled  pellicle  in  bouillon,  of  proper  alkalinity,  containing  gly- 
cerin or  carbohydrates  (cane  sugar,  lactose) ;  also  that  in  such  media  the 
formation  of  indol  and  a  subsequent  return  to  an  alkaline  reaction  may  be 
observed. 

"  As  already  stated,  Sanarelli  isolated  from  Seine  water  a  considerable 
number  of  vibrios,  and  among  them  four — viz. :  one  from  St.  Cloud,  Point- 
du-Jour,  Gennevilliers  No.  5,  and  Versailles  (Seine),  which  after  twenty-four 
hours  gave  a  distinct  indol  reaction  and  were  more  or  less  pathogenic  for 
guinea-pigs  (the  one  from  St.  Cloud  was  also  pathogenic  for  pigeons}.  Ivan- 
off  (1893)  describes  a  vibrio  which  he  isolated  from  the  faeces  of  a  patient  with 


568  PATHOGENIC   SPIRILLA. 

typhoid  fever.  But  as  the  discharges  had  been  mixed  with  Berlin  hydrant 
water,  Ivanoff  admits  the  possibility  that  his  vibrio  came  from  this  water. 
It  closely  resembles  the  cholera  vibrio,  but  is  distinguished  by  its  colonies  in 
gelatin  plates,  which,  at  the  end  of  twenty -four  to  thirty-six  hours,  in  place 
of  the  usual  coarse  granulation  of  cholera  colonies  shows  a  distinct  formation 
of  filaments.  Morphologically  the  vibrio  is  distinguished  by  a  decided  ten- 
dency to  preserve  the  spiral  form,  and  especially  by  its  size.  Celli  and  San- 
tori  (1893)  describe  a  Vibrio  romanus,  which  they  isolated  from  twelve 
undoubted  cases  of  cholera.  This  does  not  give  the  indol  reaction,  is  not 
pathogenic  for  animals,  and  does  not  grow  in  bouillon  or  agar  at  37°  C. 
This  is  considered  by  the  authors  named  an  atypical  variety  of  the  cholera 
vibrio,  especially  as  the  distinguishing  characters  did  not  prove  to  be  perma- 
nent. After  eight  months'  cultivation  the  cultures  gave  the  indol  reaction,  but 
the  pathogenic  power  was  still  almost  absent.  Recently  Chantemesse  (1894) 
has  described  a  vibrio  which  he  found  in  the  spring  of  1894  during  the  chol- 
era epidemic  at  Lisbon.  This  differed  in  many  particulars  from  the  genuine 
cholera  vibrio,  resembling  more  closely  the  vibrio  of  Finkler-Prior.  As  in 
the  Lisbon  epidemic,  with  a  large  number  taken  sick,  only  one  death  occurred, 
and  in  view  of  the  results  of  the  bacteriological  examination,  Chantemesse 
supposes  this  to  have  been  an  epidemic  of  cholera  nostras.  Finally,  Pfuhl 
(1894)  found  a  vibrio  in  the  north  harbor  of  Berlin  which  from  its  growth  in 
gelatin  and  pathogenesis  for  pigeons  he  believes  to  be  identical  with  Vibrio 
Metschnikovi." 

To  the  list  of  vibrios  above  referred  to  as  resembling  more  or  less 
closely  the  cholera  spirillum  we  must  add  those  described  by  Cun- 
ningham (1894)  and  obtained  by  him  from  the  discharges  of  cholera 
patients.  He  has  described  "  thirteen  distinct  forms  obtained  from 
cases  of  cholera  and  one  of  non-choleraic  origin." 

Pfeiffer  and  Issaeff  (1894),  in  a  recent  publication,  report  that  they 
have  found  a  sensitive  test  for  the  differentiation  of  these  vibrios  in 
the  specific  character  of  cholera  immunity.  They  found  that  guinea- 
pigs  which  were  immunized  against  cholera  infection  have  a  lasting 
immunity,  and  that  the  serum  of  such  immunized  animals  has  a 
specific  action  in  protecting  against  infection  by  genuine  cholera  vib- 
rios only,  while  for  other  species  it  has  no  action  different  from  that 
of  the  blood  serum  of  normal  animals.  In  all  cases  where  the  cholera 
serum  acted  specifically  the  vibrios  were  promptly  destroyed,  while 
in  cases  where  this  specific  action  was  absent  the  injected  vibrios 
multiplied  rapidly  and  caused  the  death  of  the  animal.  By  means  of 
this  method  the  vibrios  isolated  from  water — the  phosphorescent 
vibrios  of  Dunbar,  Vibrio  Danubicus,  Cholera  Massanah — are  shown 
to  be  distinct  species,  while  the  vibrio  of  Ivanoff  behaves  like  the 
genuine  cholera  vibrio.  In  a  subsequent  paper  Pfeiffer  reports  the 
interesting  fact  that  a  trace  of  highly  active  cholera  serum,  added  to 
a  culture  of  the  cholera  spirillum,  when  injected  into  the  peritoneal 
cavity  of  a  guinea-pig,  within  a  surprisingly  brief  time  causes  the 
destruction  of  the  cholera  vibrios ;  whereas  no  such  effect  is  produced 
upon  other  species.  A  similar  destruction  occurs  when  cholera  vib- 
rios are  injected  into  the  abdominal  cavity  of  immunized  guinea- 


PATHOGENIC   SPIRILLA.  569 

pigs.  The  researches  of  Dunbar  (1894)  indicate  that  Pfeiffer's  test 
is  not  so  reliable  as  he  supposed ;  and  also  that  phosphorescence  can- 
not be  relied  upon  for  distinguishing  similar  water  bacteria  from 
genuine  cholera  vibrios.  Rumpel  has  reported  the  fact  that  two  un- 
doubted cultures  of  the  cholera  spirillum,  from  different  sources,  after 
being  passed  through  pigeons  and  cultivated  for  some  time  in  arti- 
ficial media,  showed  phosphorescence.  One  of  these  cultures  was  ob- 
tained originally  from  the  discharges  of  Dr.  Oergel,  who  was  a  vic- 
tim to  cholera  from  laboratory  infection  (case  reported  by  Reincke,  in 
the  Deutsche  medicinisclie  Woclienschrift,  No.  41,  1894).  Another 
case  of  supposed  laboratory  infection,  in  which  recovery  occurred,  is 
reported  by  Lazarus,  in  the  Berliner  medicinische  Wochenschrift, 
1893,  page  1,241. 

That  cholera  vibrios  may  be  present  in  the  alimentary  canal  of 
healthy  individuals  without  giving  rise  to  any  symptoms  of  ill-health 
appears  to  be  demonstrated.  In  support  of  this  conclusion  we  quote 
as  follows  from  a  recent  paper  by  Abel  and  Claussen : 

"  In  Wehlau  (East  Prussia),  in  the  autumn  of  1894,  seven  cases  of 
cholera  occurred  about  the  same  time.  The  members  of  the  family 
were  at  once  isolated  and  their  faeces  examined  almost  daily.  Of 
especial  interest  were  seventeen  individuals  who  belonged  to  families 
in  which  three  fatal  cases  occurred.  Of  these  seventeen  persons,  who 
were  not  sick  at  all  or  only  had  for  a  brief  time  a  diarrhoea,  thirteen 
had  cholera  vibrios  in  their  discharges  for  a  considerable  time.  As 
the  table  shows,  many  of  these  comma  bacilli  were  not  found  in  dis- 
charges every  day,  but  were  obtained  again  after  being  absent"  (in 
the  cultures)  "  for  a  day  or  two." 

Abel  and  Claussen  (1895),  as  a  result  of  very  extended  experi- 
ments, arrive  at  the  conclusion  that  cholera  vibrios  in  faaces  as  a  rule 
do  not  survive  longer  than  twenty  days,  and  often  cannot  be  ob- 
tained after  two  or  three  days;  exceptionally  they  were  obtained  in 
cultures  at  the  end  of  thirty  days — Karlinsky  and  Dunbar  have  re- 
ported finding  them  at  the  end  of  fifty-two  days  and  four  months. 
Karlinsky  (1895)  has  also  reported  that  upon  woollen  and  linen  goods, 
cotton  batting  and  wool,  which  were  soaked  in  the  discharges  of 
cholera  patients  and  preserved  from  drying  by  being  wrapped  in 
waxed  paper,  the  cholera  vibrio  retained  its  vitality  for  from  twelve 
to  two  hundred  and  seventeen  days. 

The  researches  of  Kasansky  (1895)  show  that  the  cholera  spiril- 
lum is  not  destroyed  by  alow  temperature  (—30  C.)  and  that  it 
even  resists  repeated  freezing  and  thawing — three  or  four  times. 

Behring  and  Ransom  (1895)  as  a  result  of  an  extended  experi- 
mental research,  arrive  at  the  conclusion  that  cholera  cultures  from 
which  the  bacteria  have  been  removed  have  specific  toxic  properties, 
40 


570  PATHOGENIC   SPIRILLA. 

and  cause  symptoms  similar  to  those  which  result  from  the  intro- 
duction into  guinea-pigs  of  the  living  bacteria ;  that  from  these  fil- 
tered cultures  a  solid  substance  can  be  obtained  having  the  same 
toxic  properties,  and  that  from  susceptible  animals  which  have  been 
treated  with  this  toxic  substance  a  serum  can  be  obtained  which  is 
active  not  only  against  the  cholera  poison,  but  against  the  cholera 
vibrio.  These  results  support  those  previously  reached  by  other 
bacteriologists  and  lead  to  the  hope  that  a  specific  treatment  of  the 
disease  may  be  successfully  employed.  The  results  obtained  by 
Haffkine  in  India  are  favorable  to  the  view  that  his  method  of  prophy- 
laxis, by  the  subcutaneous  injection  of  virulent  cholera  cultures,  has 
a  real  value. 


PLATE  IX. 

FIG.  1.— Bacillus  diphtherise  (Klebs-Loffler)  from  culture  on  blood  serum. 
Stained  with  Loffler's  solution  of  methylene  blue.  X  1,000.  Photomicro- 
graph by  oil  lamp.  (Borden.) 

FIG.  2.— Micrococcus  gonorrhoeas  in  urethral  pus.  Stained  with  Loffler's 
solution  of  methylene  blue.  X  1,000.  Photomicrograph  by  oil  lamp 
(Borden.) 

FIG.  3. — Bacillus  tuberculosis  in  sputum.  X  1,000.  Photomicrograph 
by  oil  lamp.  (Borden.) 

FIG.  4. --Bacillus  typhi abdominalis,  from  agar culture.  X  1,000.  Photo- 
micrograph by  oil  lamp.  (Borden.) 

FIG.  5. — Streptococcus  pyogenes  (longus).  X  1,000.  Photomicrograph 
made  at  the  Army  Medical  Museum  by  sunlight.  (Gray.) 

FIG.  6. — Bacillus  mallei.  X  1,000.  Photomicrograph  made  at  the  Army 
Medical  Museum  by  sunlight.  (Gray.) 


PLATE  IX. 

STERNBERG'S  BACTERIOLOGY 


v 

- 


Fig.    2. 


FiK.     3. 


Fig.    4. 


rv-LM  / 


PATHOGENIC  BACTERIA. 


XVI. 

BACTERIA  IN  INFECTIOUS  DISEASES. 

IN  the  present  chapter  we  shall  give  a  brief  account  of  the  re- 
searches which  have  been  made  relating  to  the  presence  of  bacteria 
in  various  infectious  diseases  of  man  and  the  lower  animals,  and  in 
localized  infections  which  have  been  supposed,  on  more  or  less  satis- 
factory evidence,  to  be  due  to  their  presence.  For  convenience  of 
reference  we  shall  arrange  these  diseases  in  alphabetical  order. 

ABSCESSES. 

The  bacteria  principally  concerned  in  the  causation  of  acute  abscesses  are : 
Staphylococcus  pyogenes  aureus  (No.  1),  Staphylococcus  pyogenes  albus 
(No.  2),  and  Streptococcus  pyogenes  (No.  5).  For  details,  see  Sec.  IV.  The 
following  species  have  also  been  found  in  acute  abscesses,  sometimes  in 
pure  culture  :  Staphylococcus  pyogenes  citreus  (No.  3),  Micrococcus  tetra- 
genus  (No.  18),  Micrococcus  pneumonia)  crouposje  (No.  8),  Bacillus  coli 
communis  (No.  89),  Bacillus  typhi  abdominalis  (No.  46),  Bacillus  pyogenes 
fcetidus  (No.  72)  obtained  from  an  abscess  near  the  anus  by  Passet,  Bacil- 
lus pyogenes  fcetidus  liquefaciens,  from  a  brain  abscess  following  otitis  media, 
by  Lanz.  Passet  also  found  in  two  abscesses  out  of  thirty-three  examined  a 
micrococcus  named  by  him  Micrococcus  cereus  albus,  and  in  a  single  case 
his  Micrococcus  cereus  flavus. 

Recent  researches  show  that  next  to  the  micrococci  mentioned  above  as 
commonly  found  in  the  pus  of  acute  abscesses  (Nos.  1,  2,  5).  The  micrococ- 
cus of  croupous  pneumonia,  the  colon  bacillus,  and  the  bacillus  of  typhoid 
fever  are  most  frequently  present — the  last  mentioned  in  abscesses  follow- 
ing typhoid  fever.  In  the  so-called  "cold"  abscesses,  due  to  tubercular  in- 
fection of  glands,  the  tubercle  bacillus  is  usually  the  only  microorganism 
present. 

See  also  Bubo,  Mastitis,  Otitis  Media. 

ACNE. 

Hodara  (1894)  finds  in  the  pustules  of  acne  a  small  bacillus  (No.  175)  which 
he  believes  to  be  the  cause  of  the  disease.  It  is  said  to  be  found  at  the  base 
and  central  portion  of  the  comedones,  while  cocci  and  flask-shaped  bacilli 
are  found  in  the  superficial  portion.  In  pseudo-acne  pustules  this  bacillus 
was  not  found. 

ACNE  CONTAGIOSA  OF  HORSES. 

Dieckerhoff  and  Grawitz  believe  the  cause  of '  *  acne  contagiosa  "  in  horses 
to  be  a  bacillus  described  by  them  (No.  141). 


572  BACTERIA  IX  INFECTIOUS  DISEASES. 


ALOPECIA. 

Robinson  (1888)  claims  to  have  found,  in  sections  from  the  diseased  skin 
in  a  case  of  alopecia  areata,  micrococci  having-  a  diameter  of  about  0.8  /*,  usu- 
ally united  in  pairs  and  associated  in  zooglcea  masses.  They  were  located 
for  the  most  part  in  the  lymph  spaces  of  the  central  portion  o'f  the  chorium. 
They  stained  with  the  usual  aniline  colors  and  also  by  Gram's  method.  No 
culture  or  inoculation  experiments  were  made. 

Kasauli  (1889)  obtained  from  the  margins  of  the  affected  patches  in  alope- 
cia areata  a  bacillus  about  two  to  three  times  as  long  as  broad,  and  which 
formed  spores.  It  was  attached  to  hail's  withdrawn  from  the  diseased 
patches,  and  was  easily  cultivated  in  various  media. 

Yaillard  and  Vincent  (1890),  in  a  form  of  alopecia  resembling  favus,  ob- 
tained by  cultivation,  from  hairs  pulled  out  from  the  diseased  patches,  a  mi- 
crococcus  ;  this  was  also  found  in  the  hair  follicles  in  stained  sections.  The 
diameter  of  this  micrococcus  was  about  1  n ;  it  was  easily  stained  with  the 
aniline  colors  and  by  Gram's  method ;  grew  in  nutrient  gelatin,  causing 
liquefaction  ;  did  not  grow  well  upon  potato ;  was  pathogenic  for  mice. 
When  applied  to  the  surface  of  the  body  of  guinea-pigs  or  rabbits,  by  rub- 
bing, alopecia  resulted  similar  to  that  in  the  cases  from  which  the  micrococ- 
cus was  first  obtained. 

Hollborn  (1895)  thinks  it  probable  that  alopecia  areata  is  due  to  a  micro- 
scopic fungus  described  by  him,  which  bears  some  resemblance  to  Trichophy- 
ton  tonsurans. 

Elliott  (1895)  believes  that  the  most  frequent  cause  of  alopecia  praematura 
is  some  form  or  grade  of  eczema  seborrhoicum.  See  Eczema. 

ANGINA. 

Although  the  pus  cocci  are  frequently  found  in  the  secretions  from  the 
mouth,  nares,  and  fauces  of  healthy  persons,  there  can  be  but  little  doubt 
that  they  are  concerned  in  the  etiology  of  angina,  and  of  catarrhal  or  pseudo- 
diphtheritic  inflammations  of  mucous  membranes  elsewhere. 

Dornberger  (1894)  reports  that  in  fortv-five  per  cent  of  the  healthy  indi- 
viduals examined  streptococci  were  found.  In  78.9  per  cent  of  the  cases  of 
angina  Streptococcus  longus  was  found,  but  never  in  pure  cultures ;  in  aii- 
gina  phlegmonosa  Streptococcus  brevis  was  present ;  in  seven  cases  of  acute 
catarrhal  angina  streptococci  were  found  five  times,  and  in  chronic  catarrhal 
angina  in  one-half  the  cases. 

Plaut  (1894)  in  five  cases  of  severe  angina  found  Miller's  bacillus  in  large 
numbers  in  the  exudate  in  the  fauces,  and  believes  that  it  was  the  cause  of 
the  inflammation  of  the  mucous  membrane. 

Goldschneider  (1893)  found  in  the  angina  of  scarlet  fever  streptococci 
only  in  seven  cases,  and  staphylococci  alone  in  fourteen.  No  difference  was 
observed  in  the  exudate  in  the  cases  belonging  to  the  two  groups,  but  tlu> 
streptococcus  angina  was  more  severe  and  ran  a  more  protracted  ccnu>«; 
(average  duration  12.6  days).  In  eight  cases  streptococci  and  staphylococci 
were  associated— these  had  an  average  duration  of  thirteen  days. 

Booker  (1892)  found  streptococci  in  the  angina  of  scarlet  fever  and 
measles,  associated  in  some  cases  with  staphylococci. 

ANTHRAX. 

Due  to  the  presence  of  Bacillus  anthracis  (No.  45)  in  the  blood 
and  tissues  of  infected  animals — or  in  malignant  pustule  and  in 
"  wool-sorters'  disease  "  in  man. 


BACTERIA  IN  INFECTIOUS  DISEASES.  573 


APPENDICITIS. 

Hodenpyl  (1893)  in  ten  cases  of  appendicitis  in  which  a  bacterio- 
logical examination  was  made  found  Bacillus  coli  communis  in  pure 
culture,  and  in  one  case  the  same  bacillus  associated  with  Strepto- 
coccus pyogenes.  Including  his  own  cases  with  twenty-four  re- 
corded by  other  investigators  the  colon  bacillus  was  the  only  micro- 
organism present  in  thirty-two  out  of  the  thirty-five  cases. 

ARTHRITIS. 

In  arthritis  following  pneumonia  the  Micrococcus  pneumonia? 
crou posse  has  been  found  in  pure  culture  by  several  bacteriologists — 
Boulloche,  Schwartz,  Picque  and  Veillon,  Brunner.  In  gonorrhoeal 
arthritis  the  gonococcus  has  been  found  by  Bordoni-Uffreduzzi,  Pal- 
tauf,  Lindemann,  Neisser,  and  others.  Manley  (1894)  saj's:  "In  the 
most  virulent  cases  which  have  come  under  my  own  care  the  as- 
pirated fluid  was  found  to  contain  no  gonococci ;  while  in  other  cases 
which  ran  a  mild  course,  it  was  said  that  the  gonococcus  and  some- 
times the  diplococcus  were  seen  in  large  numbers." 

In  suppurative  arthritis  following  scarlet  fever  streptococci  have 
been  found  in  pus  from  the  affected  joints  by  Babes,  Kankin,  Len- 
harz,  and  by  Bellingham  Smith  (1895). 

BERI-BERI. 

Lacerda  (1887)  claims  to  have  demonstrated  the  presence  of  cocci,  some- 
times united  in  chains,  in  the  blood  and  tissues  of  persons  affected  with  beri- 
beri, and  also  to  have  produced  in  rabbits,  by  inoculation  with  his  cultures, 
certain  symptoms  resembling  those  which  characterize  this  disease. 

Pekelharing  and  Winkler  (1887)  have  also  obtained  by  cultivation,  from 
the  blood  of  patients  with  beri-beri,  various  forms  of  bacteria,  but  princi- 
pally cocci ;  these  are  described  as  being  usually  associated  in  pairs  or  in  ir- 
regular groups,  as  forming  a  milk-white  mass  upon  agar,  and  as  liquefying 
gelatin.  According  to  the  authors  named,  injection  into  rabbits  of  cultures 
of  this  coccus  gave  rise  to  multiple  nerve  degeneration,  such  as  is  seen  in 
cases  of  beri-beri  in  man. 

Eykmann  (1888)  failed  to  obtain  cultures  from  the  blood  of  patients  with 
beri-beri,  but  demonstrated  the  presence  of  slender  bacilli  similar  to  those 
which  Pekelharing  and  Winkler  encountered  in  some  of  their  cases.  These 
failed  to  grow  in  the  usual  culture  media. 

In  his  latest  communication  upon  the  subject  Pekelharing  says  that  in 
twelve  cases  out  of  fifteen  he  obtained  cultures  of  micrococci,  and  bacilli  in 
three  out  of  fifteen.  From  his  inoculation  experiments  he  concludes  that 
the  micrococci  found  are  the  cause  of  the  morbid  phenomena  which  charac- 
terize the  disease. 

When  in  Eio  de  Janeiro  (1887)  the  writer  collected  blood  from  the  finger 
from  four  typical  cases  of  beri-beri,  selected  by  Dr.  Lacerda,  and  introduced 
it  into  the  usual  culture  media.  The  result  of  this  experiment  was  negative, 
agreeing  in  this  regard  with  the  results  obtained  by  Eykmann. 

Musso  and  Morelli  (1893)  report  that  they  obtained  from  the  blood,  sub- 


574  BACTERIA   IX  INFECTIOUS  DISEASES. 

cutaneous  oedema,  ascitic  fluid,  etc.,  of  two  persons  who  died  of  beri-beri,  a 
micrococcus,  which,  when  injected  into  rabbits,  caused  their  death  in  from 
forty  days  to  four  months,  with  symptoms  similar  to  those  of  beri-beri. 
Their  micrococcus  is  from  0.8  to  2.4  //  in  diameter ;  in  pairs  or  in  chains  ; 
stains  by  Gram's  method  and  liquefies  gelatin- 

BISKRA  BUTTON. 
See  Micrococcus  of  Heydenreich  (No.  26). 

BRONCHITIS. 

Lumnitzer  (1888)  obtained  from  the  sputum  of  a  patient  with  putrid  bron- 
chitis a  bacillus  which  proved  to  be  pathogenic  for  mice  and  for  rabbits,  and 
the  cultures  of  which  gave  off  a  characteristic  odor,  similar  to  that  of  the 
putrid  bronchial  secretion  in  his  patient  (No.  112). 

Picohini  (1889),  in  three  cases  of  "  croupous  bronchitis,"  made  culture  ex- 
periments and  isolated  three  different  micrococci ;  one  developed  upon  nutri- 
ent gelatin  as  a  grayish- white  mass  and  did  not  liquefy  ;  one  as  a  reddish- 
gray  mass,  also  non-liquefying  ;  the  third  form  was  always  associated  with 
these  two. 

Bernabei  (1895)  has  found  the  bacillus  of  Lumnitzer  in  a  number  of  cases 
of  putrid  bronchitis,  and  believes  it  to  be  the  cause  of  the  disease.  Alfieri 
(1894)  has  also  reported  a  case  in  which  a  bacillus  was  found  which  appears 
to  be  the  same.  Hitzig  (1895)  obtained  two  bacilli  resembling  the  colon  bacil- 
lus f  rom  a  case  of  putrid  bronchitis  investigated  by  him. 

BRONCHO-PNEUMONIA. 

Netter  (1892)  has  made  a  bacteriological  study  of  95  fatal  cases  of 
broncho-pneumonia,  53  adults  and  42  children.  Of  the  adult  cases 
39  gave  a  pure  culture  of  a  single  species,  which  in  15  was  the  mi- 
crococcus of  croupous  pneumonia,  in  12  Streptococcus  pyogenes,  in  !> 
Fried  lander's  bacillus,  in  3  staphylococci.  In  14  cases  of  mixed  in- 
fection the  micrococcus  of  pneumonia  and  staphylococci  were  found 
in  5  ;  the  pneumonia  coccus  and  streptococci  in  3  ;  the  pneumonia 
coccus  with  Friedlander's  bacillus  in  2  ;  pneumonia  cocci,  strepto- 
cocci, and  staphylococci  in  1.  In  42  cases  in  children  25  were  simple 
and  17  mixed  infection;  in  10  pneumonia  cocci  only,  in  8  streptococci, 
in  5  staphylococci,  in  2  Friedlander's  bacillus.  In  the  mixed  infec- 
tions pneumonia  cocci  and  streptococci  in  5,  streptococci  and  staphylo- 
cocci in  5,  streptococci  and  Friedlander's  bacillus  in  3  ;  pneumonia 
cocci,  streptococci,  and  staphylococci  in  2,  pneumonia  cocci  and 
staphylococci  in  1,  pneumonia  cocci  and  Friedlander's  bacillus  in  1. 
In  broncho-pneumonia  following  epidemic  influenza  (8  cases)  the 
pneumonia  coccus  was  found  in  1,  streptococci  in  1,  Friedlander's 
bacillus  in  2,  pneumonia  coccus  and  streptococcus  in  1,  streptococcus 
and  staphylococcus  in  1. 

BUBO. 
Hoffa  (1886)  in  2^  cases  of  inguinal  bubo  examined  found  Staphy- 


BACTERIA  IN   INFECTIOUS   DISEASES.  575 

lococcus  pyogenes  aureus  in  10,  Staphylococcus  pyogenes  albus  in  9, 
and  Staphylococcus  pyogenes  citreus  in  3. 

Later  researches  indicate  that,  as  a  rule,  the  pus  cocci  are  not 
present  in  the  pus  from  an  unopened  inguinal  bubo  following  chan- 
croid. In  this  regard  Ducrey,  Krefting,  and  Spietschka  are  in  ac- 
cord. The  last-named  author  also  arrives  at  the  conclusion  that  the 
streptobacillus  found  in  chancroidal  ulcers  is  not  present  in  the  pus 
of  unopened  buboes,  and  that  this  is  not  virulent.  Inoculation  ex- 
periments with  such  pus  gave  a  negative  result,  and  the  most  careful 
microscopical  investigation  failed  to  reveal  the  presence  of  micro- 
organisms of  any  kind.  Cheinisse  (1894)  also  had  a  negative  result 
from  inoculations  with  the  pus  from  buboes  except  in  one  case  in 
which  the  bacillus  of  Ducrey  was,  by  exception,  demonstrated  to  be 
present.  He  finds  that  while  the  pus  from  unopened  buboes  is 
usually  sterile  it  sometimes  contains  the  ordinary  pyogenic  micro- 
cocci. 

BUBONIC  PLAGUE. 

The  bacillus  found  by  Kitasato  (1894)  and  by  Yersin  (1894)  in  the 
contents  of  the  buboes  and  in  the  blood  of  infected  animals  is  no 
doubt  the  cause  of  this  infectious  malady  (see  No.  166). 

CARCINOMA. 

Various  bacteria  have  occasionally  been  found  in  carcinomatous  growths, 
and  especially  in  those  which  have  undergone  ulceration ;  but  that  any  one 
of  these  bears  an  etiological  relation  to  such  malignant  tumors  has  not  been 
demonstrated. 

CEREBRO-SPINAL  MENINGITIS. 

Various  microorganisms  have  been  found  by  bacteriologists  in  the 
exudate  of  cerebro-spinal  meningitis,  and  there  seems  to  be  but  little 
doubt  that  the  meningeal  inflammation  is  due  to  their  presence,  as 
the  bacteria  usually  found  are  pathogenic  for  certain  of  the  lower 
animals,  and  when  introduced  into  a  serous  cavity  they  give  rise  to 
a  fibrinous  or  purulent  inflammatory  process.  The  researches  of 
Weichselbaum,  Netter,  and  others  show  that  Micrococcus  pneu- 
monia crouposa3  ("  diplococcus  pneumonia  ")  is  the  microorganism 
most  frequently  found,  and  next  to  this  the  Diplococcus  intercel- 
lularis  meningitidis  of  Weichselbaum.  Streptococcus  pyogenes  has 
also  been  found  in  a  certain  proportion  of  the  cases — four  out  of 
twenty-five  cases  of  purulent  meningitis  studied  by  Netter. 

Bonome,  in  a  series  of  cases  studied  by  him,  obtained  a  micro- 
coccus  closely  resembling  Micrococcus  pneumonia  crouposa3,  but 


576  BACTERIA  IN  INFECTIOUS  DISEASES. 

which  he  believes  not  to  be  identical  with  it  (see  Micrococcus  of 
Bonome,  No.  40). 

Jaeger  (1894)  from  a  study  of  the  literature  arrives  at  the  conclu- 
sion that  in  from  sixty  to  seventy  per  cent  of  the  recorded  cases  of 
idiopathic  cerebro-spinal  meningitis  the  pneumonia  coccus  ("  Diplo- 
coccus  lanceolatus")  has  been  found.  His  own  researches  lead  him 
to  believe  that  the  "  diplococcus  intercellularis  "  of  Weichselbaum  is 
the  cause  of  genuine  epidemic  meningitis,  and  that  the  pneumonia 
coccus  may  be  present  also  as  a  secondary  infection.  Sporadic  cases 
may  be  due  to  streptococcus  infection,  to  tubercular  infection,  or  to 
pneumococcus  infection.  In  meningitis  secondary  to  middle-ear  dis- 
ease the  pneumonia  coccus  is  the  usual  infectious  agent.  Sherer 
(1894)  reports  three  cases  of  leptomeningitis  purulenta  in  nursing  in- 
fants in  which  the  Bacillus  coli  communis  was  found  in  pure  cultures. 
The  infection  is  supposed  to  have  occurred  by  bathing  the  infants  in 
water  contaminated  by  their  own  discharges.  In  a  later  communi- 
cation (1895)  the  same  author  gives  an  account  of  an  epidemic  of 
cerebro-spinal  meningitis  among  soldiers  in  which  the  infectious 
agent  was  Diplococcus  intercellularis  meningitidis  (No.  9).  This 
diplococcus  was  found  in  the  nasal  secretions  of  the  infected  individ- 
uals during  life,  as  well  as  in  the  exudate  from  the  inflamed  men- 
inges,  obtained  post  mortem.  Centanni  (1893)  has  described  "a  new 
microorganism  of  meningitis"  under  the  name  Bacillus  aerogenes 
meningitidis  (No.  181). 

CHALAZION. 

Deyl  (1893)  was  unable  to  cause  the  development  of  chalazion  by  introduc- 
ing1 a  culture  of  staphylococci  into  the  mouths  of  the  Meibomian  glands  in 
man  and  rabbits.  In  fifteen  cases  of  incipient  sty  in  which  he  made  bacterio- 
logical examinations,  he  found  a  bacillus  which  he  believes  to  be  concerned 
in  the  etiology  of  this  localized  infection. 

Landwehr  (1894)  found  in  one  case  almost  a  pure  culture  of  Micrococcus 
tetragenus.  He  arrives  at  the  conclusion  that  in  a  certain  proportion  of  tbe 
cases  the  tubercle  bacillus  is  the  etiological  agent,  but  admits  that  this  has  not 
been  demonstrated,  and  that  inoculation  experiments  in  susceptible  animals 
with  the  contents  of  the  sty  have  always  given  negative  results. 

CHANCROID. 

Ducrey,  in  an  extended  research  (1890),  was  not  able  to  cultivate 
any  specific  microorganism  from  the  pus  of  soft  chancres,  or  of 
buboes  resulting  from  these  ulcers.  Various  common  microorgan- 
isms were  obtained  in  cultures  from  the  chancroidal  ulcers,  but  a 
negative  result  was  obtained  in  his  cultures  from  the  pus  of  buboes. 
In  pustules  developed  upon  the  arm  from  the  inoculation  of  chan- 
croidal virus  he  found  constantly  a  bacillus  which  did  not  grow  in 
artificial  cultures.  This  was  about  1.48  /*  long  and  0.5  /*  thick,  with 


BACTERIA   IN   INFECTIOUS    DISEASES.  577 

round  ends.  It  was  readily  stained  with  a  solution  of  fuchsin,  but 
not  by  Gram's  method. 

Unna,  in  1892,  reported  that  in  five  cases  of  chancroid  he  had 
found  a  strepto-bacillus,  which  on  account  of  its  numbers  and  situa- 
tion in  the  tissues  involved  would  probably  prove  to  be  the  specific 
cause  of  this  localized  infection.  Quinquaud  (1892)  confirmed  Unna 
as  to  the  presence  of  a  strepto-bacillus,  but  neither  of  the  bacteriolo- 
gists named  succeeded  in  obtaining  this  bacillus  in  cultures.  Since 
this  date  numerous  papers  have  been  published  with  reference  to  the 
presence  of  this  and  other  bacilli  in  chancroidal  ulcers.  In  his  latest 
communication  (1895)  Unna  maintains  that  the  bacillus  of  Ducrey 
is  in  fact  identical  with  his  strepto-bacillus.  He  says  that  the  pres- 
ence of  this  bacillus  has  now  been  demonstrated  in  more  than  one 
hundred  cases;  and,  on  the  other  hand,  it  has  never  been  found  in 
pus  from  other  sources.  It  is  readily  stained  by  Gram's  method,  and 
this  serves  to  distinguish  it  from  the  only  similar  strepto-bacillus — 
which  is  often  found  in  serpinginous  chancroid  and  especially  about 
the  margins.  Unna  asserts  that  this  strepto-bacillus  is  constantly 
present,  that  it  is  the  only  microorganism  in  the  chancroidal  tissue, 
and  that  it  has  not  been  found  elsewhere.  He  therefore  feels  justified 
in  concluding  that  it  is  the  specific  etiological  agent,  although  it  has 
not  as  yet  been  obtained  in  pure  cultures. 

Spietschka  (1894)  also  reports  the  presence  of  strepto-bacilli  in 
chancroidal  ulcers,  and  says  that  the  bacilli  seen  by  him  correspond 
with  those  found  by  Unna  and  later  by  Peterson,  as  to  size,  arrange- 
ment, and  location,  but  that  they  have  rounded  ends  and  a  constric- 
tion in  the  middle  and  do  not  stain  by  Gram's  method — i.e.,  they 
correspond  with  the  bacillus  described  by  Ducrey.  Peterson  (1894) 
has  also  found  the  bacillus  of  Ducrey  in  his  cases,  in  St.  Petersburg, 
and  thinks  there  can  be  no  doubt  that  it  is  the  specific  etiological 
agent.  Krefting  has  never  failed  to  find  this  bacillus  in  chancroidal 
virus,  and  in  inoculations  made  with  such  virus  the  quicker  and 
more  intense  the  result  the  more  numerous  were  the  bacilli  found  to  be. 

For  the  staining  of  cover-glass  preparations  Krefting  recommends 
the  methylene  blue  solution  of  Sahli :  Aqua  destillata,  40  cubic  centi- 
metres; saturated  aqueous  solution  of  methylene  blue,  24  cubic  centi- 
metres; solution  of  borax  (five  per  cent)  16  cubic  centimetres.  Ac- 
cording to  Krefting  the  bacilli  are  from  1.5  to  2  v-  long,  and  from  0.5 
to  1  /j.  broad.  Ducrey  describes  his  bacilli  as  short,  thick  rods,  with 
round  ends,  and  at  times  a  slight  constriction  in  the  middle.  Unna 
describes  his  bacilli  as  short  rods,  l£  to  2  v-  long  and  £  /*  broad,  ar- 
ranged in  chains  of  four  to  ten  elements.  These  chains  are  con- 
stantly '  found  in  the  lymph  spaces,  between  the  cells — never  in  the 
leucocytes  or  blood-vessels. 


578  BACTERIA   IN   INFECTIOUS   DISEASES. 

CHOLERA  ASIATICA. 

The  etiological  relation  of  Koch's  "comma  bacillus"  to  cholera  is 
now  generally  accepted  by  bacteriologists  and  pathologists.  But 
recent  researches  show  that  Spirillum  cholera  Asiatics  (No.  180) 
does  not  always  present  identical  biological  characters  when  obtained 
from  different  cases  of  epidemic  cholera;  and  that  very  similar 
spirilla  are  sometimes  found  as  saprophytes  in  river  water,  and  in 
the  alvine  discharges  of  healthy  persons.  We  call  attention  to  the 
fact  that  these  cholera-like  spirilla  have  for  the  most  part  been  found 
in  Europe,  where  epidemic  cholera  has  been  widely  diffused  during 
the  past  few  years.  It  is  probable  that  a  considerable  number  of 
them,  at  least,  are  saprophytic  varieties  of  the  genuine  cholera  spiril- 
lum. 

CHOLERA  INFANTUM. 

The  researches  of  Booker  and  of  Jeffries  do  not  support  the  idea 
that  cholera  infantum  is  due  to  the  presence  of  a  specific  micro- 
organism in  the  intestine,  but  rather  that  the  symptoms  are  due  to 
the  absorption  of  toxic  products  formed  in  the  alimentary  canal,  or 
in  the  child's  food  before  it  is  ingested,  as  a  result  of  the  multiplica- 
tion and  ferment  action  of  various  microorganisms,  and  especially  of 
certain  putrefactive  bacteria.  The  common  putrefactive  bacillus, 
Proteus  vulgaris,  and  other  species  nearly  related  to  this,  were  found 
by  Booker  in  a  considerable  proportion  of  his  cases,  and  he  is  dis- 
posed to  believe  that  these  putrefactive  bacteria  play  an  important 
part  in  the  development  of  the  morbid  phenomena  which  characterize 
the  disease.  Jeffries,  after  reviewing  the  various  theories  which 
have  been  advanced  in  explanation  of  the  etiology  of  cholera  in- 
fantum, says :  "  Bacteria  I  believe  to  be  at  the  bottom  of  the  disease 
— that  is,  rule  bacteria  out  of  all  foods  and  the  alimentary  canal,  and 
summer  diarrhoea  would  cease  to  be."  Upon  another  page  of  bis 
memoir  he  says :  "  Passing  a  step  further,  the  symptoms,  pathology, 
and  etiology  of  summer  diarrhoea  stand  in  close  relationship  with 
the  group  of  symptoms  first  clearly  brought  to  light  by  Panum  as 
putrid  infection.  The  animals  poisoned  by  the  injection  of  putrid 
fluids,  sterile  or  not,  sicken  and  die  with  two  variable  groups  of 
symptoms:  one  referable  to  the  nervous  system,  the  other  to  the  in- 
testines, diarrhoea  being  a  prominent  symptom,  and  the  autopsy  re- 
vealing inflammatory  changes  in  the  intestine." 

CHOLERA    NOSTRAS. 

What  has  been  said  above  with  reference  to  cholera  infantum 
applies  as  well  to  cholera  uostras.  This  has  not  been  shown  to  be 


BACTERIA  IN  INFECTIOUS  DISEASES.  579 

due  to  the  presence  in  the  alimentary  canal  of  a  particular  micro- 
organism ;  but  it  can  scarcely  be  doubted  that  the  morbid  phenomena 
are  induced  by  the  development  of  toxic  substances  as  a  result  of  the 
ferment  action  of  various  species  of  bacteria. 

Finkler  and  Prior  (1884)  obtained  from  the  fa?ces  of  patients  with 
cholera  nostras  a  spirillum  which  they  supposed  to  be  the  specific 
cause  of  this  disease,  but  subsequent  researches  have  not  confirmed 
their  conclusion.  Thus,  in  seven  cases  studied  by  bacteriological 
methods  in  Koch's  laboratory  during  the  years  1885,  1886,  and  1887, 
the  spirillum  of  Finkler  and  Prior  was  not  found  in  a  single  instance 
(Frank). 

In  an  epidemic  of  cholera  nostras,  in  which  five  cases  out  of  seven 
proved  fatal,  Carp  (1893)  was  not  able  to  find  spirilla  in  the  dis- 
charges from  the  bowels,  or  in  the  drinking-water  to  which  the  out- 
break was  ascribed.  The  drinking-water  was,  however,  found  to  be 
very  bad  and  to  contain  "fa3ces  bacilli."  Kirchner  (1892)  in  sixteen 
cases  of  cholera  nostras  examined  failed  to  find  the  spirillum  of 
Finkler  and  Prior — in  two  cases  he  found  a  spirillum  which  failed  to 
grow  in  gelatin  plates  and  in  three  a  streptococcus. 

Ruete  and  Enoch  (1894)  in  a  fatal  case  of  cholera  nostras  obtained 
from  the  small  intestine  a  spirillum  which  they  identified  as  that  of 
Finkler  and  Prior.  The  authors  named  state  that  researches  made  in 
the  laboratories  of  Koch,  Hueppe,  and  Baumgarten  show  that  "  Mil- 
ler's bacillus,"  which  is  occasionally  found  in  the  mouths  of  healthy 
persons,  is  identical  with  the  spirillum  of  Finkler  and  Prior. 

Grube  (1887)  has  reported  a  fatal  case  of  cholera  nostras  in  which 
this  spirillum  was  present  in  the  intestine,  and  Lustig  (1887)  reports 
two  fatal  cases  of  cholera  in  which  it  was  associated  with  Koch's 
"comma  bacillus."  In  view  of  the  facts  stated,  and  of  the  patho- 
genic properties  of  this  spirillum,  we  have  the  same  reasons  for  sup- 
posing that  it  is  the  cause  of  those  cases  of  cholera  nostras  in  which 
it  is  found,  as  for  assuming  the  etiological  relation  of  the  Spirillum 
cholera  Asiaticse.  But  it  is  evident  that  cholera  nostras  is  not  a 
specific  disease  due  to  the  pathogenic  action  of  a  single  microorgan- 
ism. On  the  other  hand,  the  experimental  evidence  indicates  that  in 
this  disease,  and  in  cholera  infantum,  summer  diarrhoea,  and  other 
gastro-intestinal  disorders,  the  toxic  products  developed  by  various 
bacteria  may  give  rise  to  the  symptoms  characterizing  these  diseases. 

CONJUNCTIVITIS. 

The  various  forms  of  conjunctivitis  have  been  ascribed  to  the 
specific  action  of  bacteria.  That  this  is  true  as  regards  gonorrhceal 
ophthalmia  is  now  generally  admitted,  and  there  is  some  reason  to 


580  BACTERIA   IN   INFECTIOUS   DISEASES. 

believe  that  the  bacillus  discovered  by  Koch  and  studied  by  Kartulis 
(see  Bacillus  of  Kartulis)  is  the  cause  of  one  form  of  "  Egyptian  ca- 
tarrhal  conjunctivitis."  The  non-infectious  forms  of  conjunctivitis 
can  scarcely  be  supposed  to  be  due  to  the  action  of  specific  micro- 
organisms; but  it  is  probable  that  an  inflammation  resulting  from 
any  cause,  such  as  a  chemical  or  mechanical  irritant,  may  be  ag- 
gravated and  become  chronic  as  a  result  of  the  presence  of  various 
microorganisms,  and  especially  of  the  pyogenic  micrococci. 

Kain  (1892)  from  a  case  of  croupous  conjunctivitis  obtained  a 
bacillus  which  when  introduced  into  the  conjunctival  sacs  of  rabbits 
is  said  to  have  caused  a  purulent,  membranous  inflammation. 

Wilbrand,  Sanger,  and  Staelin  (1893)  have  investigated  an  epi- 
demic of  conjunctivitis  in  patients  at  their  eye  clinic  in  Hamburg 
with  the  following  results:  "With  a  high  degree  of  probability  we 
may  conclude  that  a  diplococcus  plays  the  principal  role  in  the  eti- 
ology of  this  epidemic.  As  already  indicated,  these  diplococci  in 
smear  preparations  resemble  the  gonococcus  of  Neisser,  and  were  rec- 
ognized as  such  by  all  unprejudiced  and  competent  observers  among 
our  colleagues;  but  this  decision  soon  proved  to  be  erroneous,  inas- 
much as  inoculations  in  the  urethra  of  two  men  with  the  secretion 
from  two  severe  cases,  at  the  outset  of  the  epidemic,  gave  a  com- 
pletely negative  result.  Further,  the  diplococci  lying  in  the  cells  are 
distinguished  from  gonococci  by  the  fact  that  they  stain  by  Gram's 
method,  and  that  they  show  an  evident  growth  in  nutrient  gelatin." 
In  their  culture  experiments  the  authors  named  obtained  four  differ- 
ent diplococci — viz.,  Micrococcus  flavus  desidens,  a  common,  non- 
pathogenic  species,  found  in  the  air  and  in  water ;  Micrococcus  sub- 
flavus,  a  micrococcus  closely  resembling  the  "  trachomacoccus  "  of 
Michel ;  and  a  diplococcus  which  they  believe  to  be  new  and  which 
proved  to  be  pathogenic  for  animals.  They  are  not,  however,  cer- 
tain whether  any  one  of  these  corresponds  with  the  diplococcus  found 
in  the  pus  cells,  and  which,  unlike  the  gonococcus,  does  not  stain  by 
Gram's  method.  Certain  cases  were  characterized  by  the  presence 
of  bacilli  and  the  absence  of  diplococci.  The  bacillus  found  in  these 
cases,  within  the  pus  cells,  corresponded  with  the  bacillus  first  dis- 
covered by  Koch  in  cases  of  Egyptian  ophthalmia  (Bacillus  of  Kartu- 
lis, No.  138). 

4 'CORN-STALK  DISEASE"  IN  CATTLE. 

Billings  in  1888  ascribed  this  disease  to  a  bacillus,  and  Burrill  (1889)  sub- 
sequently described  "a  bacterial  disease  of  corn."  According  to  Billings 
the  bacillus  of  Burrill  is  identical  with  that  to  which  he  ascribes  the  "  corn- 
stalk disease"  of  cattle.  Pyle  (1893)  says:  "Comparing  the  two  germs  in 
cultivation,  I  doubt  their  identity,  though  I  recognize  their  great  similarity 
in  developing.  On  slides  they  present  no  marked  difference,  to  me,  in  appear- 


BACTERIA   IN  INFECTIOUS   DISEASES.  581 

aiice."  Pyle  further  says:  "Dr.  Billings  lias  made  many  successful  experi- 
ments in  connection  with  this  disease.  He  has  succeeded  in  causing-  death  in 
susceptible  animals  by  feeding  corn-stalk  leaves  and  tops  of  corn  supposed  to 
be  diseased  with  the  '  bacterial  disease  of  corn '  which  Dr.  Burrill  has  so 
completely  described.  From  animals  thus  destroyed  Dr.  Billings  obtained 
pure  cultures  of  the  *  corn-stalk  disease.'  With  these  cultures  he  destroyed 
susceptible  animals  by  inoculation." 

CORYZA. 

Hajek  found  in  the  secretions  of  acute  nasal  catarrh  a  large  diplococcus, 
called  by  him  "  Diplococcus  coryzae,"  and  probably  identical  with  the  diplo- 
coccus previously  obtained  by  Klebs  from  the  same  source.  This  was  most 
abundant  during  the  early  stage  of  the  disease,  when  the  secretion  from  the 
nasal  mucous  membrane  was  thin  and  abundant ;  later  various  other  micro- 
organisms were  encountered  in  greater  numbers,  and  among  them  Fried- 
lander's  bacillus.  There  is  no  satisfactory  evidence  that  the  diplococcus  of 
Hajek  or  any  other  known  bacteria  are  directly  concerned  in  the  etiology  of 
this  affection.  To  what  extent  chronic  nasal  catarrh  is  due  to  the  action  of 
microorganisms  is  also  uncertain,  but  it  appears  probable  that  they  play  an 
important  part  in  maintaining  such  inflammations  ;  and  in  ozaena  the  offen- 
sive odor  of  the  nasal  secretions  is  no  doubt  due  to  the  presence  of  certain 
bacteria,  whatever  may  be  the  relation  of  these  to  the  morbid  process  which 
gives  rise  to  the  chronic  discharge.  (See  Bacillus  foetidus  ozsena?  of  Hajek.) 

CYSTITIS. 

The  extensive  researches  which  have  been  made  during  the  past 
few  years  show  that  the  presence  of  bacteria  in  the  healthy  bladder 
does  not  induce  cystitis,  but  that  when  the  mucous  membrane  is  in- 
jured by  mechanical  violence,  or  by  the  presence  of  a  foreign  body, 
cystitis  is  likely  to  result  from  the  introduction  of  bacteria,  and  that 
the  Bacillus  coli  communis  is  most  frequently  concerned  in  the  de- 
velopment of  chronic  inflammation  of  the  bladder. 

In  the  extended  researches  of  Rovsing — thirty  cases  of  cystitis — 
the  following  results  were  obtained :  In  one  case  diagnosed  as  cysti- 
tis no  bacteria  were  found ;  in  three  cases  culture  experiments  gave  a 
negative  result,  but  the  tubercle  bacillus  was  found  in  the  urine  by 
microscopical  examination — in  these  cases  the  urine  was  strongly 
acid;  in  twenty-six  cases  the  urine  was  ammoniacal,  and  in  all  of 
these  bacteria  were  found — usually  but  a  single  species.  All  of  these 
grew  in  the  usual  culture  media  except  the  tubercle  bacillus,  which 
in  two  cases  was  associated  with  some  other  species,  and  all  pro- 
duced alkaline  fermentation  in  sterile  urine  when  added  to  it  in 
pure  cultures.  The  following  species  were  found:  Tubercle  bacil- 
lus, Staphylococcus  pyogenes  aureus,  Staphylococcus  pyogenes  albus, 
Staphylococcus  pyogenes  citreus,  Streptococcus  pyogenes  urese  (n. 
sp.),  Diplococcus  pyogenes  urese  (n.  sp.),  .Coccobacillus  pyogenes 
ureaa  (n.  sp.),  Micrococcus  pyogenes  ureae  flavus  (n.  sp.),  Diplococ- 
cus urea3  trifoliatus  (n.  sp.),  Streptococcus  urese  rugosus  (n.  sp.), 
Diplococcus  urea3  (n.  sp.),  Coccobacteria  urese  (n.  sp.). 


582  BACTERIA   IN   INFECTIOUS   DISEASES. 

Pure  cultures  of  all  of  these  species  introduced  into  the  bladder 
of  rabbits  failed  to  induce  cystitis,  even  when  injected  in  consider- 
able quantities.  But  when  retention  of  urine  was  effected  artificially 
for  six  to  twelve  hours,  allowing  time  for  ammoniacal  fermentation 
to  occur,  cystitis  was  developed.  When  the  pyogenic  species  were 
introduced  under  these  circumstances,  a  suppurative  inflammation  of 
the  mucous  membrane  occurred ;  the  non-pyogenic  species  caused  a 
catarrhal  cystitis.  Rovsing  records  the  important  fact,  as  bearing 
upon  the  etiology  of  cystitis,  that  in  twenty  of  the  cases  examined 
the  bladder  had  been  invaded  by  the  finger  or  by  instruments  prior 
to  the  development  of  cystitis. 

Lundstrom  (1800)  isolated  from  alkaline  urine  obtained  from  pa- 
tients with  cystitis  two  species  of  staphylococci — Staphylococcus 
urese  candidus  and  Staphylococcus  urese  liquef  aciens ;  from  albu- 
minous, acid  urine  he  obtained  Streptococcus  pyogenes.  Krogius  ob- 
tained from  the  urine  of  individuals  suffering  from  cystitis  a  bacillus 
which  he  calls  Urobacillus  liquefaciens  septicus.  Schnitzler  (ISiMi) 
found  the  same  bacillus,  or  one  very  similar  to  it,  in  thirteen  out  of 
twenty  cases  of  purulent  cystitis  examined  by  him.  In  eight  of  these 
cases  it  was  obtained  from  the  urine  in  pure  cultures,  and  in  five  it 
was  associated  with  other  bacteria.  In  twelve  of  these  twenty  cases 
the  cystitis  resulted  directly  from  catheterization ;  in  the  others  it 
occurred  in  individuals  suffering  from  stricture  or  from  calculus. 

When  cultures  of  this  bacillus  were  injected  into  a  vein  in  rab- 
bits, the  animals  died  in  from  three  to  eight  days,  and  in  every  in- 
stance an  intense  nephritis  was  observed  at  the  autopsy — twice  with 
the  formation  of  small  abscesses.  The  bacillus  was  found  in  the 
blood  and  the  organs  generally.  Injections  into  the  bladder  of  rab- 
bits almost  always  gave  rise  to  a  severe  purulent  cystitis — large  rab- 
bits were  selected  and  great  care  taken  not  to  injure  the  mucous 
membrane  of  the  bladder.  Schnitzler  was  not  able  to  induce  cystitis 
in  rabbits  by  injecting  in  the  same  way  considerable  quantities  of  a 
culture  of  Staphylococcus  pyogenes  aureus. 

Guyon  (1888)  did  not  succeed  in  inducing  cystitis  by  the  injection 
of  pure  cultures  of  various  microorganisms  into  the  bladder,  unless 
he  at  the  same  time  produced  an  artificial  retention  of  urine.  His 
experimental  results  are  therefore  in  accord  with  those  of  Rovsing, 
who  found  that  without  mechanical  injury,  or  artificial  retention 
until  ammoniacal  fermentation  had  occurred,  no  results  followed  his 
injections  into  the  bladder. 

According  to  Schmidt  and  Aschoff  (1893)  subsequent  researches 
indicate  that  some  of  the  species  described  by  Rovsing  as  being  new 
are  in  fact  varieties  of  Bacillus  coli  communis. 

The  identification  of  the  Bacillus  pyogenes  of  Albarran  and  Halle 


BACTERIA  IN  INFECTIOUS  DISEASES.  583 

(Bacterie  septique  of  Clado)  with  Bacillus  coli  communis  was  first 
made  by  Krogius  (1891),  and  about  the  same  time  by  Achard  and 
Renault. 

In  twelve  cases  of  cystitis,  six  of  which  were  complicated  with 
ascending  nephritis,  Krogius  demonstrated  the  presence  of  Bacillus 
coli  communis,  and  showed  that  in  its  growth  in  culture  media  it 
corresponded  with  the  Bacillus  pyogenes  of  previous  authors.  In  a 
second  communication  Krogius  states  that  in  twenty-two  cases  of 
cystitis  studied  by  him  he  obtained  Bacillus  coli  communis  sixteen 
times,  and  of  these  fourteen  times  in  pure  culture.  He  also  calls  at- 
tention to  the  fact  that  in  those  cases  where  no  other  microorganism 
was  associated  with  the  colon  bacillus  the  urine  was  always  acid — 
a  statement  which  is  sustained  by  the  subsequent  researches  of 
Schmidt  and  Aschoff.  He  also  gives  details  with  reference  to  the 
pleomorphism  of  this  bacillus  and  differences  in  the  appearance  of 
gelatin  cultures  from  different  sources,  the  growth  being  sometimes 
opaque  and  sometimes  transparent.  When  cultures  of  this  bacillus 
were  injected  into  the  bladder  of  rabbits  and  retained  by  ligating  the 
urethra,  an  intense  cystitis  was  developed  in  from  twenty  to  thirty 
hours.  Injections  into  the  ureter  gave  a  result  similar  to  that  subse- 
quently reported  by  Schmidt  and  Aschoff.  The  animal  died  in  about 
two  days,  and  pyelitis,  together  with  more  or  less  necrosis  of  the 
renal  epithelium,  was  found  at  the  autopsy.  Reblaub  (1892)  ob- 
tained Bacillus  coli  communis  in  pure  culture  in  six  out  of  sixteen 
cases  of  cystitis  examined. 

In  their  latest  publication  Achard  and  Renault  arrive  at  the  con- 
cluson  that  there  are  some  differences  between  their  "  urobacillus " 
and  Bacillus  coli  communis,  which  they  state  as  follows: 

1.  Upon  most  media,  especially  upon  malt  agar,  the  growth  is 
more  luxuriant. 

2.  Cultures  of  the  urobacillus  upon  potato  appear  grayish  white, 
very  luxuriant,  and  have  many  gas  bubbles. 

3.  The  urobacillus  develops  much  gas  even  in  gelatin  and  agar 
cultures  containing  little  sugar. 

Morelle  (1892)  and  Denys  (1892)  in  their  bacteriological  re- 
searches obtained  from  numerous  cases  of  cystitis  a  bacillus  which 
they  identified  with  Bacillus  lactis  aerogenes  of  Escherich.  But  the 
last-mentioned  author  has  since  stated  that  this  bacillus  presents 
varieties  which  cannot  be  distinguished  from  the  typical  cultures  of 
Bacillus  coli  communis. 

The  recent  researches  referred  to  having  shown  that  Bacillus  coli 
communis  is  very  commonly  present  in  the  urine  in  cases  of  cystitis, 
and  often  in  pure  cultures,  its  etiological  relation  to  the  disease  in 
question  seems  probable  ;  and  this  view  is  further  sustained  by  exper- 


584  BACTERIA   IN  INFECTIOUS  DISEASES. 

iments  upon  animals  and  by  the  demonstrated  fact  that  retention  of 
urine  per  se  does  not  give  rise  to  inflammation  of  the  bladder.  But 
this  is  not  the  only  microorganism  which  is  capable  of  causing  a 
cystitis  when  introduced  into  a  bladder  which  has  suffered  some  kind 
of  mechanical  injury  or  has  been  subjected  to  the  action  of  chemical 
irritants  contained  in  the  urine.  The  researches  of  Krogius,  Schnitz- 
ler,  and  of  Schmidt  and  Aschoff  show  that  next  to  the  colon  ba- 
cillus the  microorganisms  most  commonly  found  in  cases  of  cystitis 
and  of  pyelonephritis  is  a  proteus  (Proteus  vulgaris?). 

At  the  date  of  the  publication  of  the  monograph  of  Schmidt  and 
Aschoff  the  Bacillus  coli  communis  had  been  found  in  pure  culture  in 
sixty  cases  of  cystitis,  and  the  proteus  in  thirteen  cases. 

An  important  point  to  be  kept  in  view  is  the  fact  that  when  Ba- 
cillus coli  communis  is  found  in  the  urine  in  pure  culture,  this  fluid 
is  more  or  less  acid,  as  the  bacillus  in  question  does  not  give  rise  to 
alkaline  fermentation,  at  least  not  under  the  conditions  found  in  the 
bladder  and  in  the  absence  of  retention.  But  when  proteus  is  present 
the  urine  is  almost  always  ammoniacal. 

DENGUE. 

McLaughlin  (1886)  has  claimed  to  find  micrococci  in  the  blood  of  patients 
suffering  from  dengue.  No  satisfactory  evidence  of  their  etiological  relation 
has  been  presented,  and  his  observations  have  not  yet  been  confirmed  by 
other  investigators. 

DENTAL  CARIES. 

The  extended  researches  of  Miller  lead  him  to  the  conclusion  that  dental 
caries  is  due  to  various  microorganisms  described  by  him.  In  a  paper  pub- 
lished in  1894  his  conclusions  are  formulated  as  follows  : 

"1.  In  infectious  processes  in  the  pulp,  almost  without  exception,  we 
have  a  mixed  infection,  and  cocci  and  bacilli  are  found  with  about  equal  fre- 
quency ;  somewhat  less  frequently,  long  slender  filaments  and  spiral  forms 
are  encountered.  At  times  very  peculiar  forms  are  seen  ;  spore-bearing 
rods  and  filaments  are  occasionally  encountered. 

"2.  The  microscopical  examination  of  cover-glass  preparations  justifies 
the  view  that  micrococci  are  especially  concerned  in  the  production  of  pus. 

"3.  The  bacteria  make  their  way  to  the  pulp  principally  through  the 
carious  dentine,  and  a  thin  layer  of  hard  dentine  does  not  protect  it  with  cer- 
tainty from  infection.  Infection  of  the  pulp  through  the  blood-vessels  may 
IM-  possible  in  certain  cases,  but  has  not  been  demonstrated. 

"4.  The  pulp  is  predisposed  to  infection  by  the  action  of  products  formed 
in  the  carious  dentine  (acids-ptomaines). 

"5.  In  disease  of  the  pulp  bacteria  are  chiefly  concerned  which  cannot 
be  cultivated. 

"(>.  Various  bacilli  which  can  be  cultivated  have  been  found  in  di- 
pulp,  but  for  the  most  part  they  are  non-pathogenic. 

"7.  The  typical  pyogenic  cocci,  Streptococcus  pyogenes  aureus  and  allms, 
and  Streptococcus  pyogenes,  are  seldom  found  in  pus  from  the  pulp, 
the  contrary,  various  cocci  are  found,  ('specially  a  group  of  nearly  related 
snecies,  which  cause  pus-formation  in  mice.     This  question  has  not  yet  been 
cleared  up. 


BACTERIA   IN   INFECTIOUS   DISEASES.  585 

"8.  A  micrococcus  which  I  could  identify  with  the  micrococcus  of  spu- 
tum septicaemia,  i.e.,  the  pneumococcus,  in  spite  of  numerous  experiments 
on  animals,  I  have  as  yet  failed  to  find.  At  best  we  can  only  speak  of  a  va- 
riety of  the  pneumococcus. 

"9.  The  action  of  the  pulp  cocci  is  greatly  increased  by  putrefactive  proc- 
esses. A  putrid  pulp,  whether  bacteria  may  be  obtained  from  it  in  pure 
cultures  or  not,  is  always  a  dangerous  infectious  material. 

' '  10.  Putrid  decomposition  of  the  tooth  pulp  is  caused  by  various  bacteria, 
and  the  putrid  products  are  not  always  the  same.  In  addition  to  gaseous 
products  (NHs,  SH2)  there  are  various  other  substances,  the  nature  of  which 
has  not  been  determined." 

DIARRHOEA. 

In  the  green  diarrhoea  of  infants  Lesage  obtained  a  bacillus  (No.  106) 
which  he  supposed  to  be  the  cause  of  the  malady.  His  bacillus  is  probably 
identical  with  Bacillus  fluorescens  non-liquefaciens.  Vogler  (1893)  obtained 
from  a  diarrhceal  stool  a  vibrio  different  from  that  of  cholera,  and  which 
was  not  pathogenic  for  guinea-pigs.  We  have  referred  to  the  researches  of 
Booker  and  of  Jeffries  under  the  heading  "Cholera  Iiifantum."  Bajinsky 
(1894)  agrees  with  Fliigge  in  believing  that  the  toxins  produced  by  bacteria 
are  the  usual  cause  of  summer  diarrhoea  in  children,  a  view  in  which  we 
fully  concur.  But  there  is  no  reason  to  suppose  that  any  particular  micro- 
organism of  this  class  has  a  specific  role  in  the  etiology  of  affections  of  this 
class.  Probably  bacilli  of  the  colon  group  and  of  the  prpteus  group  are  more 
frequently  than  any  others  responsible  for  gastro-intestinal  troubles  in  chil- 
dren. They  are  very  widely  distributed  and  multiply  with  great  rapidity 
under  favorable  temperature  conditions  in  milk  or  other  articles  of  liquid 
food. 

-       DIARRHCEA   (INFECTIOUS)    IN  CALVES. 

Jensen  (1892)  has  investigated  a  fatal  infectious  disease  of  calves,  charac- 
terized by  diarrhoea,  etc.,  and  concludes  that  it  is  due  to  a  bacillus  which  cor- 
responds with  Bacillus  coli  communis  in  all  respects,  except  in  its  increased 
virulence.  In  the  contents  of  the  intestine  of  calves  which  have  recently 
succumbed  to  the  malady,  the  bacillus  is  found  almost  in  pure  culture  ;  also 
in  the  inflamed  mucous  membrane,  in  the  hyperaemic  mesenteric  glands,  and 
in  the  blood  and  various  organs. 

Calves  fed  with  a  culture  of  this  bacillus  invariably  died  within  two  or  three 
clays,  and  the  bacilli  were  found  in  almost  pure  culture  in  the  contents  of  the 
intestine,  and  in  great  numbers  in  the  blood  and  organs.  The  subcutaneous 
injection  of  four  cubic  centimetres  of  a  bouillon  culture  caused  fatal  septi- 
caemia. 

DIPHTHERIA. 

The  Klebs-Loffler  bacillus  (No.  47)  is  now  generally  recognized  as 
the  specific  infectious  agent  in  diphtheria. 

DIPHTHERIA  IN   CALVES. 
Due  to  Loffler's  Bacillus  diphtheriae  vitulorum  (No.  50). 

DIPHTHERIA  IN  PIGEONS. 

Due  to  Lb'ffler's  Bacillus  diphtheriae  columbrarum  (No.  49). 
41 


586  BACTERIA   IN  INFECTIOUS  DISEASES. 

DOGS,    INFECTIOUS  DISEASES  OF. 

According  to  Schantyr  (1893)  the  so-called  "distemper"  in  dogs  includes 
three  different  infectious  diseases — "abdominal  typhus,  typhoid,  and  genuine 
distemper."  In  several  cases  of  so-called  distemper  ("  staupe  ")  he  obtained  a 
bacillus,  previously  described  by  Semmer,  which  reproduced  the  disease  when 
inoculated  into  healthy  animals.  This  bacillus  closely  resembles  Bacillus 
typhi  abdominalis,  and  is  perhaps  identical  with  it,  but  its  virulence  for  ani- 
mals is  greater.  In  typhoid  of  dogs  he  found  in  the  blood  and  various  or- 
gans small  bacilli,  which  stained  by  Gram's  method  and  also  closely  resem- 
bled the  bacillus  of  typhoid  fever  in  man.  Young  dogs  died  within  a  short 
time  when  inoculated  with  cultures  of  this  bacillus.  In  thirteen  cases  of 
genuine  distemper,  Schantyr  found  in  the  blood  and  organs  a  great  number 
of  bacilli,  from  1  to  2  p>  long,  mostly  associated  in  groups,  which  he  was  not 
able  to  cultivate  in  the  usual  media.  Once  only  he  obtained  an  agar  culture 
and  from  this  a  culture  on  blood  serum.  Two  out  of  three  dogs  inoculated 
with  this  culture  died  of  distemper. 

Galli-Valerio  (1895)  has  reviewed  the  literature  relating  to  the  etiology  of 
distemper  in  dogs,  and  reports  the  results  of  his  own  investigations.  He 
found  constantly  in  young  dogs  which  succumbed  to  the  disease  an  oval  ba- 
cillus, 1.25  to  2. 5  n  long,  and  0.31  /*  thick.  This  was  present  in  the  lungs, 
the  brain,  and  the  spinal  marrow,  but  never  in  the  blood.  In  gelatin  cul- 
tures gas  bubbles  appeared  along  the  line  of  puncture  within  twenty-four 
hours,  and  a  small,  white,  wax-like  point  was  developed  on  the  surface  ;  later 
along  and  narrow  funnel  was  seen,  but  this  did  not  contain  liquefied  gelatin. 
The  bacillus  was  readily  stained  with  the  aniline  colors  and  by  Grain's 
method.  A  young  dog,  five  months  old,  succumbed  to  a  subcutaneous  inocu- 
lation, at  the  end  of  eighteen  days,  with  all  the  symptoms  of  distemper.  And 
the  bacillus  was  recovered  from  the  pustules,  the  lungs,  brain,  and  spinal 
marrow. 

DYSENTERY. 

While  the  evidence  seems  to  support  the  view  that  certain  cases  of  dysen- 
tery are  due  to  the  presence  of  the  amoeba  coli,  this  parasite  is  not  found  in 
others.  Thus  in  twenty  cases  studied  by  Maggiori  (1893)  it  was  only  found  in 
one ;  and  in  an  epidemic  of  dysentery  in  Japan,  studied  by  Ogata,  amoebae 
were  not  found  in  the  discharges.  In  the  epidemic  observed  oy  Maggiori, 
three  deaths  occurred  out  of  two  thousand  and  one  cases  ;  the  duration  of  the 
disease  was  from  six  to  twelve  days.  The  bacteriological  examination  dem- 
onstrated the  presence  of  Bacillus  coli  communis  in  great  numbers,  and  in 
most  of  the  cases  of  Proteus  vulgaris,  but  not  in  great  abundance ;  in  six 
cases  Bacillus  fluorescens  liquefaciens  was  found  ;  in  two  Staphylocoecus 

Syogenes  aureus  ;  in  one  Staphylococcus  pyogenes  albus  ;  in  fiveout  of  eleven 
acillus  pyocyaneus  in  small  numbers.  The  colon  bacillus  and  Bacillus  py<>- 
cyaneus  proved  to  be  very  virulent.  Ogata  found  in  the  recent  discharges,  in 
great  numbers,  fine,  short  bacilli,  which  liquefied  gelatin  and  stained  l»y 
Gram's  method.  This  bacillus  proved  to  be  pathogenic  for  animals,  and  is 
believed  by  Ogata  to  have  been  the  cause  of  the  epidemic  observed  by  him. 
Arnaud  (1894)  investigated  sixty  cases  of  tropical  dysentery,  and  arrives  at  the 
conclusion  that  it  is  due  to  a  pathogenic  variety  of  the  colon  bacillus.  I 
dogs  inoculated  in  the  rectum  with  his  cultures  of  this  bacillus  had  dysentery 
as  a  result  of  these  inoculations,  with  characteristic  ulceration  of  the  colon. 
Laveran  ((1893),  also,  only  found  amoebae  in  one  case  out  of  ten  of  "  Euro- 
pean dysentery  "  studied  by  him  A  bacillus  which  was  apparently  identical 
with  Bacillus  coli  communis  was  present  in  great  numbers.  Bertram!  and 
Baucher  (1893)  have  studied  an  epidemic  among  the  troops  stationed  at  Cher- 
bourg, and  arrive  at  the  conclusion  that  no  one  of  the  microorganisms  found 
by  them  can  be  considered  as  specific  for  the  disease.  They  found  among 


BACTERIA   IN   INFECTIOUS   DISEASES.  587 

other  bacteria  present  in  the  dysenteric  discharges  :  Bacillus  coli  communis, 
Bacillus  pyocyaneus,  Bacillus  fluorescens  liquefaciens,  Staphylococcus  pyo- 
genes aureus,  and  Bacillus  cedematis  maligni. 

ECLAMPSIA. 

G-erdes  (1892)  obtained  from  the  kidneys,  lungs,  liver,  and  blood  from  the 
aorta  of  two  fatal  cases  of  puerperal  convulsions  a  bacillus  which  he  supposed 
to  be  the  cause  of  the  disease.  Hofmeister  (1892)  has  shown  that  the  bacillus 
of  Gerdes  was,  in  fact,  the  well-known  saprophyte,  Proteus  vulgaris.  Haeg- 
ler  (1892)  in  examinations  of  the  blood  of  cases  of  puerperal  eclampsia  always 
had  a  negative  result,  but  in  the  urine  various  bacteria  were  found — in  one 
case  Proteus  vulgaris,  in  one  Micrococcus  urese,  in  one  Staphylococcus  pyo- 
genes  albus,  in  one  a  diplococcus  which  was  probably  identical  with  the  micro- 
coccus  of  croupous  pneumonia.  Doderlein  (1893)  also  failed  to  find  bacteria 
of  any  kind  in  the  blood  of  patients  with  puerperal  eclampsia — or  in  the  urine. 
Bar  and  Renon  (1894),  in  three  cases  in  which  a  bacteriological  examination 
of  the  liver  was  made  immediately  after  death,  found  in  one  Staphylococcus 
pyogenes  albus  and  aureus  ;  in  the  other  cases  cultures  from  the  liver  re- 
mained sterile.  Combemale  and  Bue  (1892)  report  that  in  four  cases,  in 
which  there  was  albuminuria,  oedema,  and  disturbance  of  vision,  a  bac- 
teriological examination  of  the  blood  demonstrated  the  presence  of  Staphy- 
lococcus pyogenes  albus,  alone  or  associated  with  Streptococcus  pyogenes 
albus. 

ECZEMA. 

Unna  and  Tommasoli  (1889)  have  described  three  species  of  micrococci 
and  six  bacilli  obtained  from  cases  of  eczema  seborrhoicum  which  they  re- 
garded as  new,  viz. :  Bacillus  liquefaciens  fluorescens  minutissimus,  Bacillus 
aureus,  Bacillus  spiriferus,  Bacillus  albicans  pateriformis,  Bacillus  ovatus 
minutissimus,  Ascobacillus  citreus,  Diplococcus  citreus  liquefaciens,  Diplo- 
coccus liquefaciens  tardus,  Diplococcus  albicans  tardus. 

Merrill  (1895)  has  made  a  bacteriological  study  of  fifty  cases  of  eczema 
seborrhoicum.  In  two  cases  the  result  was  negative,  "it  being  impossible  to 
obtain  any  growth  from  the  scales  by  any  method."  In  the  remaining  forty- 
eight  cases  bacteria  were  obtained  in  cultures,  made  at  the  room  temperature. 
"Pure  cultures  obtained  in  the  experiments  showed  three  distinct  varieties 
of  bacteria  which  may  be  designated  Nos.  1,  2,  and  3.  In  thirty-one  cases 
all  three  were  present ;  in  seven  only  Nos.  1  and  2 ;  in  two,  Nos.  1  and  3  ; 
in  five,  No.  1 ;  and  in  one,  No.  3  alone.  Staphylococcus  pyogenes  aureus 
was  also  obtained  in  one  case  and  Bacillus  fluorescens  liquefaciens  minutis- 
simus in  three.  The  bacteria  found  are  described  as  follows  : 

"  Variety  1. — Small  diplococci,  single  or  in  irregular  groups.  The  parts 
forming  each  diplococcus  are  round  or  only  slightly  oval.  The  germs  are 
aerobic,  noii -liquefying,  and  iion-chromogenic.  At  70°  F.  they  grow  rap- 
idly. On  gelatin  plates  the  deep-seated  colonies  remain  about  the  size  of  an 
ordinary  pin's  head  for  weeks.  The  superficial  colonies  are  round,  white, 
with  slightly  raised  surfaces,  and  smooth  or  somewhat  irregular  borders.  In 
its  growth  the  colony  adheres  very  nearly  to  its  circular  form.  After  the 
first  week  the  centre  begins  to  turn  darker,  and  with  increasing  age  of  the 
colony  the  whole  surface,  hitherto  smooth,  begins  to  be  wrinkled  and  the 
edges  become  irregular,  as  though  the  evaporation  of  the  water  caused  con- 
traction. At  the  end  of  three  weeks  growth  seems  to  stop,  and  the  colony 
changes  from  its  original  white  color  to  a  dusky  brown.  On  agar-agar  the 
appearances  closely  resemble  those  of  the  gelatin  colonies,  except  that  it  is 
slower  in  its  growth  and  its  surface  has  a  whiter  lustre.  On  potato  the 
growth  begins  to  be  visible  on  the  second  day.  On  the  fifth  day  it  is  cream 
white,  smooth,  raised  about  a  tenth  of  an  inch,  and  its  edges  are  irregular 


588  BACTERIA   IN   INFECTIOUS   DISEASES. 

and  scalloped.  At  this  time  it  covers  about  two-thirds  of  the  surface  of  a 
potato  stick  half  an  inch  in  diameter.  After  the  first  week  the  growth  is 
slow,  and  at  the  age  of  three  weeks  its  size  only  equals  that  of  the  iirst  week, 
but  the  colony  itself  is  shrivelled,  dried,  and  dark  in  color.  In  milk  the  cul- 
ture had  on  the  second  day  a  slight  greenish  tinge,  which,  by  the  fifth  day. 
had  disappeared.  The  upper  quarter  inch  of  the  milk  seems  slightly  thicker, 
but  no  other  change  is  visible  to  the  naked  eye. 

"  Variety  2. — In.  appearance  it  is  almost  identical  with  Variety  1,  except 
that  it  seems  more  oval  in  form.  This  diplococcus  is  aerobic,  non-liquefy- 
ing, and  chromogenic.  As  in  Variety  1,  the  ordinary  changes  of  tempera- 
ture, as  occur  from  the  rotation  of  the  seasons  of  the  year,  retard  or  acceler- 
ate the  growth  of  the  culture.  On  Petri  dishes  of  gelatin,  the  minute, 
round,  yellow  colonies  appear  on  the  third  or  the  fourth  day.  Those  on  the 
surface  grow  slowly,  are  slightly  raised,  and  have  smooth  borders.  After 
the  first  week's  growth  the  centre  shows  a  deeper  orange  color.  On  agar' 
'agar,  the  growth  is  slightly  lustrous,  thicker,  and  of  a  light  orange  color. 
On  potato,  a  deep  golden  layer  develops,  which  is  well  raised  and  has  irreg- 
ular borders.  In  milk,  this  diplococcus  grows  as  Variety  1  does,  except 
that  after  ten  days  the  upper  layer  of  the  milk  is  thickened  and  has  turned 
the  same  golden  color  mentioned.  In  stab  cultures  of  Varieties  1,  and  2  the 
growth  adheres  pretty  closely  to  the  puncture  line,  gradually  spreading 
down  it  and  over  the  surface. 

"  Variety  3. — A  bacillus  with  rounded  ends,  single,  in  pairs,  or  in  short 
or  long  chains.  It  is  aerobic  and  anaerobic,  motile,  liquefying,  and  non- 
chromogenic.  In  gelatin  tubes  a  grayish- white  growth  commences  on  the 
second  day.  In  smear  cultures  a  pit  of  liquefied  gelatin  is  formed,  and,  re- 
maining of  the  same  irregular  shape  as  the  smear,  it  gradually  deepens  and 
contains  at  the  bottom  a  whitish  sediment.  In  stab  cultures  the  resulting  pit 
is  the  shape  of  the  puncture  and  contains  the  same  white  sediment.  On  aga  r- 
agar  the  growth  is  whitish,  its  surface  raised,  without  lustre,  and  its  border 
indented. 

"  Inoculation  experiments  were  attempted  in  twelve  cases.  The  site  of 
inoculation  chosen  was  the  hairy  scalp,  or  over  the  sternum.  After  thor- 
oughly sterilizing  the  skin,  two  or  three  hairs  were  pulled  out  and  the  skin 
slightly  abraded,  as  in  vaccination  ;  portions  of  an  actively  growing  culture 
were  then  rubbed  in  with  a  sterilized  platinum  needle.  With  No.  3  two 
attempts  were  made  and  both  failed.  With  No.  1  five  attempts  were  made. 
Of  these,  one  was  a  failure.  In  the  four  others  the  edges  of  the  inoculation 
spots  began  to  grow  slightly  reddened  from  the  fourth  to  the  sixth  day,  and 
small  scales  formed  on  the  surface.  By  the  seventh  to  the  tenth  day  t lie 
spots  had  increased  in  size  and  were  covered  with  dry  white  scales.  Scales 
taken  from  these  spots  and  placed  in  suitable  culture  media  in  each  case  gave 
rise  to  pure  cultures  of  diplococcus  No.  1.  Variety  No.  2  was  used  ome. 
On  the  sixth  day  yellowish  scales  appeared  over  the  surface.  They  grew 
slightly  more  marked  on  the  tenth  aay,  and  the  lesion  then  closely  resem- 
bled certain  typical  forms  of  seborrhceic  eczema.  Diplococcus  No.  II.  was 
found  in  the  cultivations  from  these  scales.  The  last  four  inoculations  were 
made  with  both  No.  1  and  No.  2.  Of  these,  one  was  a  failure.  Another 
showed  a  small  spot  covered  with  a  few  branny  scales,  too  small  to  allow  of 
any  conclusions  being  drawn.  In  the  other  two  achange  began  on  the  fourth 
day.  The  bases  began  to  redden,  and  typical,  crumbly,  greasy  scales  began 
to  cover  the  surfaces  and  pile  up  in  the  centres.  On  the  eighth  day  the  spots 
were  an  eighth  of  an  inch  across,  and  represented  patches  of  seborrhoeic 
eczema.  Both  Nos.  1  and  2  could  be  cultivated  from  these  scales.  It  is 
possible  that  Variety  No.  2  may  have  given  the  yellow  color  to  those  seal. •-. 
as  it  was  absent  in  the  successful  cases 'using  No.  1. 

"The  result  of  the  twelve  inoculation  experiments,  therefore,  are  :  Five 
failures.  Seven  cases  in  which  definite  lesions  were  produced." 


BACTERIA   IN   INFECTIOUS   DISEASES.  589 

ECZEMA   EPIZOOTICA. 

monym. — Foot  and  mouth  disease. 

Tnis  is  an  infectious  disease  of  horned  cattle,  characterized  by  a  vesicular 
eruption  in  the  mouth  and  about  the  feet.  It  affects  also  sheep  and  pigs  and 
may  be  communicated  to  man. 

Schottelius  (1892)  has  described  a  microorganism  which  he  thinks  may 
bear  an  etiological  relation  to  the  disease.  His  inoculation  experiments  do- 
not,  however,  sustain  this  view,  inasmuch  as  the  characteristic  vesicles  were- 
never  developed  in  inoculated  calves,  and  experiments  upon  other  animals- 
gave  a  negative  result.  In  young  cattle  small  doses  (one  cubic  centimetre) 
of  a  bouillon  culture  gave  rise  to  a  slight  fever  and  loss  of  appetite,  while 
larger  doses  produced  an  intense  fever,  salivation,  and  great  debility.  But 
recovery  occurred  at  the  end  of  five  or  six  days  without  any  aphthous  erup- 
tion. Schottelius  obtained  from  the  clear  contents  of  the  vesicles  in  the. 
mouth  various  bacteria  which  he  believes  to  have  been  accidentally  present. 
After  making  a  considerable  number  of  culture  experiments  his  attention 
was  attracted  by  a  spherical  microorganism,  united  in  chains,  which  grew 
very  slowly  in  the  ordinary  culture  media.  This  he  describes  as  follows  : 

The  individual  cells  vary  greatly  in  diameter,  and  are  considerably  larger 
than  known  micrococci ;  they  are  associated  in  longer  or  shorter  chains,  and 
are  endowed  with  active  movements.  According  to  Schottelius,  they  be- 
long to  the  "  streptocyt en  "  rather  than  to  the  streptococci.  They  do  not 
stain  readily  with  methylene  blue,  but  may  be  stained  with  gentian  violet 
and  by  Gram's  method.  Development  does  not  occur  at  temperatures  below 
37°  to  39°  C.  The  most  suitable  culture  medium  was  found  to  be  bouillon  or 
glycerin  agar  to  which  formate  of  soda  had  been  added  (amount?).  Growth 
occurred  in  an  atmosphere  of  CO2  as  well  as  in  atmospheric  air.  Upon, 
plates  of  nutrient  agar — containing  glycerin  and  formate  of  soda — at  37°  C., 
very  delicate,  almost  transparent  colonies  developed  ;  they  were  of  a  pearl- 
gray  color,  with  an  irregular,  rosette-like  margin ;  in  the  course  of  several 
weeks  they  attained  a  diameter  of  one  to  one  and  one-half  millimetres. 
Upon  potato  a  scanty,  grayish-white,  dry  layer  is  developed.  Under  the 
most  favorable  conditions  the  development  was  very  slow — not  more  rapid 
than  that  of  the  tubercle  bacillus. 

Kurth  (1894)  obtained  from  the  contents  of  vesicles  on  the  udders  of  in- 
fected animals  seven  different  microorganisms,  six  of  which  were  not  con- 
stantly present,  while  the  seventh  was  found  in  great  numbers  in  all 
cases,  with  one  exception,  and  was  also  present  in  the  saliva.  This  was  a 
streptococcus,  named  by  Kurth  Streptococcus  involutus.  Pure  cultures  of 
this  streptococcus  were  rubbed  into  the  mouths  of  young  sheep  and  calves 
without  result.  Sanfelice  (1894)  in  an  extended  research  verified  the  pres- 
ence of  the  Streptococcus  involutus  of  Kurth  in  the  aphtiious  vesicles  and 
superficial  erosions  of  the  tongue  in  infected  animals.  But  his  inoculations 
of  pure  cultures  into  susceptible  animals  gave  a  negative  result,  and  he  con- 
cludes that  this  streptococcus  is  not  concerned  in  the  etiology  of  the  disease. 

Piano  and  Fiorentini  (1895)  arrive  at  the  conclusion  that  the  disease  is 
not  due  to  any  microorganism  belonging  to  the  schizomycetes ;  but  that  it  is 
probably  due  to  an  amoeboid  microorganism  which  is  found  in  the  contents 
of  the  vesicles  and  in  the  blood  of  infected  animals.  This  conclusion  is  in 
accord  with  the  views  of  Schottelius  and  of  Behla,  and  will  probably  prove  to 
be  well  founded. 

EMPYEMA. 

"A.  Frankel  (1888),  as  a  result  of  his  bacteriological  studies  in 
twelve  cases  of  empyema,  divides  the  cases  into  four  groups.  In  one 
group  of  three  cases  Streptococcus  pyogenes  was  the  only  microor- 


590  BACTERIA   IN  INFECTIOUS   DISEASES. 

ganism  obtained  in  his  cultures  or  seen  in  stained  preparations  of 
pus  from  the  pleural  cavity.  In  a  second  group  of  three  cases,  oc- 
curring in  the  course  of  a  pneumonia,  the  only  microorganism  pres- 
ent was  "  diplococcus  pneumonia? "  (Micrococcus  pneumonia?  crou- 
posa?).  The  third  group  included  four  cases  of  tubercular  emp3rema; 
in  one  of  these  tubercle  bacilli  only  were  found  in  pus  from  the 
pleural  cavity,  in  one  case  streptococci  were  found,  and  in  two  no 
microorganisms  were  found.  In  the  fourth  group  of  two  cases  the 
empyema  resulted  from  the  opening  of  an  abscess  into  the  pleural 
cavity,  and  streptococci  were  found  in  the  pus. 

Netter,  in  a  series  of  forty-six  cases  examined  by  him,  found  Mi- 
crococcus pneumonia?  crouposa?  in  fourteen.  Koplik  (1890)  found 
the  same  microorganism  in  seven  cases  examined  by  him,  and  Strep- 
tococcus pyogenes  in  two  cases. 

Weintrand  (1893)  has  reported  a  case  of  empyema  following  ty- 
phoid fever,  in  which  the  typhoid  bacillus  in  pure  culture  was  found 
in  pus  drawn  from  the  pleural  cavity  by  means  of  a  syringe. 

Prudden  (1893)  found  microorganisms  in  every  case  examined  by 
him  (twenty-four) ;  in  seven  cases  out  of  eight  Streptococcus  pyo- 
genes was  present  in  pus  obtained  from  the  pleural  cavity.  In  the 
cases  of  metapneumonic  empyema  the  germ  most  commonly  present 
(in  nine  cases  out  of  eleven)  was  the  Micrococcus  lanceolatus  (pneu- 
mococcus).  In  four  cases  of  foetid  empyema  various  bacilli  were 
found.  Staphylococcus  pyogenes  aureus  was  present  in  one  case 
only. 

Levy  (1895),  from  a  review  of  the  literature  of  the  subject  in 
connection  with  his  own  observations,  arrives  at  the  conclusion  that 
Streptococcus  pyogenes  is  the  usual  cause  of  purulent  inflammation 
of  the  pleura — found  in  sixty  per  cent  of  the  cases. 

ENDOCARDITIS. 

The  experimental  evidence  relating  to  endocarditis  is  similar  to 
that  in  cystitis.  The  injection  of  the  microorganisms  found  attached 
to  the  diseased  structures  into  the  circulation  of  lower  animals  does 
not  produce  endocarditis  unless  the  valves  have  been  previously  in- 
jured by  mechanical  violence  or  by  chemical  irritants.  If  some 
doubt  remains  among  pathologists  as  to  the  etiological  relation  of  the 
microorganisms  found,  the  serious  secondary  results  of  the  mycotic 
invasion  are  well  established.  In  a  series  of  twenty-nine  cases  stud- 
ied by  Weichselbaum  (1885-1888)  the  following  results  were  ob- 
tained :  In  eight  the  result  of  culture  experiments  and  microscopical 
examination  was  negative;  in  seven  "diplococcus  pneumonia?"  (Mi- 
crococcus pneumonia?  crouposa?)  was  found;  in  six  Streptococcus 


BACTERIA   IN   INFECTIOUS   DISEASES.  591 

pyogenes ;  in  two  Staphylococcus  pyogenes  aureus ;  in  two  Bacillus 
endocarditidis  griseus  ( Weichselbaum) ;  in  one  Micrococcus  endo- 
carditidis  rugatus  (Weichselbaum);  in  one  Bacillus  endocarditidis 
capsulatus  (Weichselbaum);  in  two  cases  a  bacillus  which  he  did 
not  succeed  in  cultivating.  For  further  details  see  the  descriptions 
of  microorganisms  referred  to. 

Howard  (1893)  reports  a  case  of  acute  ulcerative  endocarditis  in 
which  the  diphtheria  bacillus  was  present  in  pure  culture — also  ob- 
tained in  cultures  from  the  liver,  spleen,  and  kidneys.  In  a  case  of 
malignant  endocarditis  in  a  patient  with  gonorrhoea  and  gonorrhoeal 
rheumatism,  Leyden  (1893)  found  the  gonococcus  in  the  vegetations 
upon  the  valves.  Banti  (1894)  in  22  cases  examined  obtained 
Streptococcus  pyogenes  in  7,  Staphylococcus  pyogenes  aureus  in  1, 
these  two  microorganisms  associated  in  3,  the  micrococcus  of  pneu- 
monia in  8  ;  in  2  no  bacteria  were  found.  Dessy  (1894)  also  exam- 
ined 22  cases  and  had  a  negative  result  in  2.  In  the  cases  in 
which  bacteria  were  present  he  found  "  Diplococcus  lanceolatus  cap- 
sulatus "  (Micrococcus  pneumonias  crouposaa)  in  8,  Streptococcus  py- 
ogenes in  7,  Staphylococcus  pyogenes  aureus  in  1,  Staphylococcus 
pyogenes  aureus  and  Streptococcus  pyogenes  associated  in  3,  Staphy- 
lococcus pyogenes  albus  and  Diplococcus  lanceolatus  in  1. 

ENDOMETRITIS. 

La  Place  (1892)  has  reported  the  results  of  his  bacteriological  investiga- 
tions of  the  secretions  of  the  endometrium  and  cervix  uteri  in  health  and  dis- 
ease. He  found  numerous  bacteria  in  the  normal  secretions,  but  vastly  more 
in  secretions  from  the  inflamed  endometrium,  "the  superficial  exfoliating 
cells  also  containing  them."  "In  chronic  endometritis  the  secretions  con- 
tain about  as  many  infectious  organisms,  the  mucous  membrane  and  fibrous 
tissue  become  greatly  hypertrophied  under  the  continued  development  of 
these  organisms,  and  whether  this  chronic  condition  be  simple  or  gonor- 
rhceal,  we  find  the  germs  both  in  the  epithelium  and  fibrous  tissue."  The 
microorganisms  obtained  from  the  secretions  of  women  with  endocervicitis 
were  the  ordinary  pus  cocci,  Bacillus  pyocyaneus,  and  certain  other  bacteria 
designated  by  the  letters,  x,  y,  and  z. 

Wolf  (1893),  in  the  secretions  from  the  uterus  in  eight  women  suffering 
from  endometritis,  found  micrococci  to  be  most  numerous  ;  but  in  two  cases 
bacilli  were  found,  and  in  one  a  vibrio  somewhat  resembling  Koch's  "com- 
ma bacillus."  This  he  describes  under  the  name  of  Bacillus  choleroides. 

ERYSIPELAS. 

Due  to  infection  by  Streptococcus  pyogenes  (No.  5) . 

ERYTHEMA. 

Cordua  (1885)  obtained  from  a  series  of  cases  of  an  erysipelatoid  skin 
affection  of  the  fingers  and  hands,  which  he  identified  as  corresponding  with 
erythema  exudativum  multiforme  of  Hebra,  a  micrococcus  resembling 
Staphylococcus  pyogenes  albus  in  its  biological  characters,  but  which  he  de- 


592  BACTERIA   IN   INFECTIOUS   DISEASES. 

scribes  as  being  three  to  four  times  as  large.  Inoculations  in  animals  were 
without  result,  but  two  inoculations  upon  his  own  hand  produced  a  dark-red 
tumefaction  in  the  vicinity  of  the  point  of  inoculation  resembling  that  in  the 
individuals  from  whom  he  obtained  his  cultures. 

In  two  cases  of  "  polymorphous  erythema"  Haushalter  (1887)  isolated  a 
streptococcus  which  did  not  produce  an  erysipelatous  inflammation  when  in- 
oculated into  the  ear  of  rabbits,  and  which  he  supposed  to  be  a  different 
species  (?)  from  the  now  better  known  Streptococcus  pyogenes.  In  five  cases 
of  erythema  nodosum  in  children  Demme  obtained  a  bacillus  which  his  inocu- 
lation experiments  proved  to  be  pathogenic,  and  which  was  perhaps  con- 
cerned in  the  etiology  of  the  skin  affection  from  which  his  cultures  were  ob- 
tained (see  Bacillus  of  Demme,  No.  107). 

Finger  (1892)  has  reported  a  case  in  which  there  was  also  an  extensive 
diphtheritic  process  in  the  throat,  and  metastatic  abscesses  in  the  kidneys 
and  myocardium  from  which  Streptococcus  pyogenes  was  obtained  in  pure 
cultures.  In  the  erythema  papules,  also,  were  found  great  masses  of  strep- 
tococci, exclusively  in  the  blood-vessels  and  filling  the  capillaries  of  the  pap- 
illary bodies  as  if  by  an  injection  mass.  In  erysipelas  the  streptococcus  is 
not  found  in  the  blood-vessels,  but  invades  the  lymph  channels. 

FARCY  IN  CATTLE. 
See  Bacillus  of  Nocard  (No.  60). 

FISH,    INFECTIOUS   DISEASES   OF. 

See  Bacillus  piscicidus  (No.  173),  Bacillus  piscicidus  agilis  (No.  167),  Bacil- 
lus of  Emmerich  and  Weibel  (No.  169). 

FOOT  AND   MOUTH  DISEASE. 
See  Eczema  epizob'tica. 

FOWL  CHOLERA. 
Due  to  infection  by  Bacillus  septicaemias  hemorrhagicae  (No.  61). 

FURUNCULOSIS. 

Due  to  infection  by  the  ordinary  pus  cocci  (Nos.  1,  2,  5),  and  especially  by 
Staphylococcus  pyogenes  aureus. 

FROGS,  INFECTIOUS  DISEASES  OF. 
See  Bacillus  hydrophilus  fuscus,  of  Sanarelli  (No.  81). 

GANGRENE. 

When  the  vital  resistance  of  the  tissues  is  impaired  by  malnutrition  and 
pressure,  or  by  an  impaired  blood  supply  from  any  cause,  an  invasion  by 
saprophytic  bacteria  is  liable  to  occur  and  a  more  or  less  extensive  gangrene 
results.  It  is  probable  that  the  infectious  disease  known  as  ''hospital  gan- 
grene" is  due  to  common  saprophytes  which  have  attained  increased  patho- 
genic virulence  as  a  result  of  special  conditions  relating  to  their  environment 
in  suppurating  wounds.  This  has  not,  however,  been  demonstrated,  and  itn 
possible  that  the  development  of  an  epidemic  of  hospital  gangrene  is  due  to 
the  introduction  of  some  pathogenic  microorganism  different  from  those 
usually  found  in  the  secretions  of  wounds  and  which  has  the  power  of  invad- 
ing healthy  tissues  when  introduced  into  an  open  wound. 


BACTERIA  IN  INFECTIOUS  DISEASES.  593 

GAS  PHLEGMON. 

In  four  cases  of  so-called  gas  phlegmon  Frankel  (E.)  found  an  anaerobic 
bacillus  named  by  him  Bacillus  phlegmones  emphysematosse.  Cultures  of 
this  bacillus  gave  rise  to  a  similar  process  when  injected  subcutaneously 
in  guinea-pigs.  In  a  case  reported  by  Bunge  (1894)  Bacillus  coli  com- 
munis  is  believed  to  have  been  the  infectious  agent  to  which  the  develop- 
ment of  the  gas  phlegmon  was  due. 

GLANDERS. 

Due  to  infection  by  Bacillus  mallei  (No.  56). 

GONORRHOEA. 

Due  to  infection  by  Micrococcus  gonorrhoeas  (No.  6) — "Gono- 
coccus"  of  Neisser. 

GRANULOMA  FUNGOIDES   (MYCOSIS  FUNGOIDES). 

Rindfleisch  (1885)  and  Auspitz  (1885)  report  the  presence  of  streptococci  in 
the  capillary  vessels  of  the  papillary  body  and  of  the  subcutaneous  tissue  in 
the  affected  localities  in  cases  of  this  disease.  That  the  streptococcus  differs 
from  Streptococcus  pyogenes,  as  Auspitz  supposes,  has  not  been  definitely 
established. 

GROUSE  DISEASE. 

See  Bacillus  of  grouse  disease,  of  Klein  (No.  76). 

HOG  CHOLERA. 

Due  to  infection  by  a  motile  bacillus  of  the  "colon  group" — 
Bacillus  of  hog  cholera,  of  Salmon  and  Smith  (No.  63). 

HOG   ERYSIPELAS. 

Due  to  infection  by  Bacillus  erysipelatos  suis  (No.  67). 

HYDROPHOBIA. 

Notwithstanding  the  extended  researches  made,  especially  in  Pasteur's 
laboratory,  the  etiology  of  hydrophobia  still  remains  unsettled.  It  has  been 
demonstrated  by  experiment  that  the  virus  of  the  disease  is  located  in  the 
brain,  spinal  marrow,  and  nerves  of  animals  which  have  succumbed  to  the 
disease,  as  well  as  in  the  salivary  secretions  of  rabid  animals,  and  that  the 
disease  may  be  transmitted  by  intravenous  inoculation,  or  by  introducing  a 
small  quantity  of  virus  beneath  the  dura  mater,  with  greater  certainty  than 
by  subcutaneous  inoculations.  But  the  exact  nature  of  this  virus  has  not  been 
determined.  The  fact  that  a  considerable  interval  elapses  after  inoculation 
before  the  first  symptoms  are  developed  indicates  that  there  is  a  multiplica- 
tion of  the  virus  in  the  body  of  the  infected  animal ;  and  this  is  further 
shown  by  the  fact  that  after  death  the  entire  brain  and  spinal  marrow  of  the 
animal  have  a  virulence  equal  to  that  of  the  material  with  which  it  was  in- 
oculated in  the  first  instance.  The  writer's  experiments  (1887)  show  that  this 
virulence  is  neutralized  by  a  temperature  of  60°  C.  maintained  for  ten  min- 
utes—a temperature  which  is  fatal  to  all  known  pathogenic  bacteria  in  the 
absence  of  spores.  But  recent  experiments  show  that  certain  toxic  products 


594  BACTERIA   IN   INFECTIOUS   DISEASES. 

of  bacterial  growth  are  destroyed  by  the  same  temperature,  We  are,  there- 
fore, not  justified  in  assuming  that  the  morbid  phenomena  are  directly  due 
to  the  presence  of  a  living  microorganism ;  and,  indeed,  it  seems  probable, 
from  what  we  already  know,  that  the  symptoms  developed  and  the  death  of 
the  animal  are  due  to  the  action  of  a  potent  chemical  poison  of  the  class 
known  as  toxalbumins.  But,  if  this  is  true,  we  have  still  to  account  for  the 
production  of  the  toxic  albuminoid  substance,  and,  in  the  present  state  of 
knowledge,  have  no  other  way  to  explain  its  increase  in  the  body  of  the  in- 
fected animal  than  the  supposition  that  a  specific,  living  germ  is  present  in 
the  virulent  material,  the  introduction  of  which  into  the  body  of  a  suscep- 
tible animal  gives  rise  to  morbid  phenomena  characterizing1  an  attack  of 
rabies. 

Pasteur  and  his  associates  have  thus  far  failed  to  demonstrate  the  pre- 
sence of  microorganisms  in  the  virulent  tissues  of  animals  which  have  suc- 
cumbed to  an  attack  of  rabies.  Babes  has  obtained  micrococci  in  cultures 
from  the  brain  and  spinal  cord  of  rabid  animals,  and  states  in  his  article  on 
hydrophobia  in  u  Les  Bacteries"  (second  edition,  page  791)  that  pure  cultures 
of  the  second  and  third  generations  induced  rabies  in  susceptible  animals ;  but 
his  own  later  researches  do  not  appear  to  have  established  the  etiological  re- 
lation of  this  micrococcus. 

Gibier  (1884)  has  reported  the  presence  of  spherical  refractive  granules, 
resembling  micrococci,  in  the  brain  of  rabid  animals,  which  he  demonstrated 
by  rubbing  up  a  little  of  the  cerebral  substance  with  distilled  water.  As 
these  supposed  micrococci  did  not  stain  with  the  usual  aniline  colors  and 
were  not  cultivated,  it  appears  very  doubtful  whether  the  refractive  granules 
seen  were  really  microorganisms. 

Fol  (1885)  claims  to  have  demonstrated  the  presence  of  minute  cocci,  0.2  u 
in  diameter,  in  sections  of  spinal  cord  from  rabid  animals,  by  Weigert's 
method  of  staining.  The  cords  were  hardened  in  a  solution  of  bichromate 
of  potash  and  sulphate  of  copper,  colored  with  a  solution  of  hsematoxylon, 
and  decolorized  in  a  solution  of  ferrocyanide  of  potash  and  borax. 

The  writer  (1887)  has  made  similar  preparations,  carefully  following  the 
method  as  described  by  Fol,  but  was  not  able  to  demonstrate  the  presence  of 
microorganisms  in  the  numerous  sections  made.  Nor  have  the  observations 
of  Fol  been  confirmed  by  the  researches  of  other  bacteriologists  who  have 
given  their  attention  to  the  subject  since  the  publication  of  his  paper. 

With  reference  to  the  results  of  Pasteur's  protective  inoculations,  we 
may  say  that  it  is  now  pretty  generally  admitted  that  the  published  statistics 
demonstrate  the  prophylactic  value  of  the  method  as  practised  at  the  Pasteur 
Institute  in  Paris. 

ICTERUS. 

Karlinsky  (1890),  in  a  series  of  five  cases  of  "  infectious  icterus  "  attended 
with  fever,  found  in  the  blood,  during  the  height  of  the  attack,  curved 
bacilli  from  two  to  six  u  long  and  one-third  to  one  n  broad,  which  were  readily 
stained  by  the  usual  aniline  colors,  but  not  by  Gram's  method.  These  he 
did  not  succeed  in  cultivating  in  any  of  the  culture  media  usually  employed. 

Ducamp  (1890)  has  also  given  an  account  of  a  "  slight  epidemic  of  infec- 
tious icterus,"  which  he  supposes  to  have  been  due  to  microorganisms. 

In  "Weil's  disease,"  which  is  characterized  by  fever  and  icterus,  and  is 
believed  to  be  an  infectious  malady,  Jaeger  (1892)  has  obtained  a  bacillus 
which  he  considers  the  specific  infectious  agents  in  the  disease — his  Proteus 
fluorescens. 

Vincent  (1893)  in  a  case  of  icterus  with  fever,  which  ended  fatally  in 
forty-eight  hours,  obtained  cultures  of  Bacillus  coli  commuiiis  from  the  blood 
and  various  organs. 


BACTERIA   IN   INFECTIOUS   DISEASES.  595 

INFLUENZA. 

Epidemic  influenza  ("  la  grippe")  is  due  to  infection  by  the  Bacil- 
lus of  influenza  (No.  52). 

INFLUENZA  OF   HORSES. 

Dieudonne  (1892),  in  an  epidemic  of  influenza  among  horses,  found  in  the 
nasal  secretions  of  infected  animals  a  micrococcus  resembling  that  of  crqup- 
ous  pneumonia  in  man.  He  did  not  succeed  in  cultivating  this  micrococcus 
in  nutrient  gelatin.  Schutz  (1888)  had  previously  cultivated  a  streptococcus 
from  the  lymphatic  glands  of  horses  suffering  from  epidemic  influenza  (Druse 
des  Pferdes)  which  he  believes  to  be  the  specific  infectious  agent  in  this  dis- 
ease (see  Streptococcus  coryzaa  contagiosse  equorum,  No.  33). 


INSECTS,    INFECTIOUS   DISEASES   OF. 

The  infectious  disease  of  bees  known  as  "foul  brood"  is  due  to  Bacillus 
alvei  (No.  14€).  Pebrine,  an  infectious  disease  of  silkworms,  is  due  to  in- 
fection by  "  Nosema  bombycis"  (No.  25).  Another  infectious  disease  of  silk- 
worms (la  flacherie)  is  believed  by  Bechamp  to  be  due  to  infection  by  Strepto- 
coccus bombycis  (No.  24).  v.  Tubeuf  (1892)  has  obtained  from  infected 
caterpillars  of  Liparis  monacha  a  motile  bacillus — Bacillus  monachae  (No. 
178).  An  infectious  disease  of  the  "chinch  bug"  (Blissus  leucopterus)  is  be- 
lieved to  be  due  to  Micrococcus  insectorum  (No.  177). 


KERATITIS. 

Bach  (1895)  as  the  result  of  his  investigations  arrives  at  the  con- 
clusion that  Ulcus  corn 86  serpens  is  due  to  invasion  of  the  cornea  by 
microorganisms,  and  that  such  invasion  is  almost  always  secondary 
to  a  traumatism,  with  loss  of  substance.  The  most  common  infec- 
tious agents  are  the  pyogenic  staphylococci  and  Streptococcus  pyo- 
genes,  but  certain  other  bacteria  are  occasionally  concerned  in  the 
localized  infectious  process.  The  researches  of  Gasparrini,  Bassi 
(1893),  Cuenod  (1895),  and  others  indicate  that  the  "diplococcus 
pneumonia"  is  not  infrequently  concerned  in  the  etiology  of  puru- 
lent keratitis,  and  this  is  confirmed  by  the  researches  of  Uhthoff 
(1895).  The  last-named  author  investigated  50  cases  of  purulent 
keratitis  in  man  with  the  following  result :  35  were  cases  of  typical 
ulcus  corna3 ;  2  of  hypopyonkeratitis,  not  of  a  serpiginous  character ; 
3  of  keratoma  lacia;  and  4  of  panophthalmia  following  corneal  infec- 
tion. In  24  cases  of  typical  ulcus  cornse  serpens  the  diplococcus  of 
pneumonia  was  found  alone,  also  in  2  cases  of  panophthalmia ;  in  7 
cases  the  pneumonia  coccus  was  found  in  association  with  other 
microorganisms — 4  of  these  were  cases  of  ulcus  cornaB  serpens;  in 
13  cases,  4  of  which  were  typical  ulcus  cornse,  the  pneumonia  coccus 
was  not  found,  but  staphylococci  or  other  bacteria  were  present ;  in 
3  cases  of  keratomalacia  streptococci  were  found.  Loeb  (1891)  ob- 


596  BACTERIA  IN   INFECTIOUS   DISEASES. 

tained  from  a  case  of  keratomalacia  a  capsule  bacillus  resembling 
that  of  Pfeiffer,  wbich  was  pathogenic  for  mice  and  for  guinea-pigs. 
The  bacillus  of  Friedlander  has  also  been  found  by  Etienue  and  by 
Yerson  and  Gabrielides  (1894)  in  "ulcus  comas  septicum." 

LEPROSY. 

No  satisfactory  experimental  demonstration  that  the  Bacillus 
leprae  is  the  cause  of  the  disease  with  which  it  is  associated  has  yet 
been  made ;  but  there  is  very  little  doubt  among  bacteriologists  and 
pathologists  that  such  is  the  case.  For  the  facts  relating  to  its  pres- 
ence in  leprous  tissues,  its  morphology,  etc.,  the  reader  is  referred  to 
the  descriptive  account  of  Bacillus  leprse  (No.  53,  page  394). 

LEUCOCYTH^KMIA. 

Pawlowsky  (1892)  in  four  cases  of  leucocythsemia  found  i»  the  blood  a 
few  short  bacilli,  with  round  ends,  which  showed  polar  staining  (with 
methylene  blue  solution).  He  did  not  succeed  in  cultivating  them  in  the 
usual  media,  but  in  a  mixture  of  bouillon  and  blood  serum  a  granular  de- 
posit was  seen  at  the  end  of  four  days,  and  transplantation  from  this  to  gly- 
cerin-agar  (plates)  gave  colonies,  at  37°  C.,  in  three  or  four  days.  These  were 
small,  round,  and  of  a  grayish-yellow  color.  Inoculations  in  rabbits  gave  a 
negative  result. 

LUPUS. 

Due  to  infection  by  Bacillus  tuberculosis  (No.  53). 

MADURA   FOOT. 

Le  Dantec  (1894)  arrives  at  the  conclusion  that  the  variety  of  madura  foot 
in  which  the  characteristic  masses  are  black  is  probably  due  to  a  bacillus 
found  by  him  in  these  "  grains."  This  bacillus  differs  from  the  streptothnx 
previously  described  by  Vincent,  and  supposed  by  him  to  be  the  cause  of  the 
malady.  It  is  difficult  of  cultivation,  and  inoculation  experiments  in  rab- 
bits and  guinea-pigs  gave  a  negative  result. 

LYMPHANGITIS. 

Lymphangitis  of  the  extremities,  according  to  Verneuil  and  Clado,  is  due 
to  infection  by  Streptococcus  pyogenes.  Fiscner  and  Levy  (1893)  as  a  result 
of  their  investigations  arrive  at  a  different  conclusion.  In  8  cases  tlu-y 
found  Staphylococcus  pyogenes  albus  in  5,  Staphylococcus  pyogenes  am  VMS 
in  1,  Bacillus  coli  communis  in  1,  Staphylococcus  pyogenes  aureus  and 
Staphylococcus  pyogenes  albus  associated  in  1.  In  abscesses  following  lym- 
phangitis (8)  Staphylococcus  pyogenes  albus  was  found  in  4,  Streptococcus 
pyogenes  in  2,  Staphylococcus  pyogenes  albus  and  Staphylococcus  pyogrm  s 
aureus  associated  in  1 ;  Staphylococcus  pyogenes  albus  and  Streptococcus 
pyogenes  in  1. 

MALARIA. 

Klebs  and  Tommasi-Crudeli,  as  a  result  of  researches  made  by  then  i  in 
the  vicinity  of  Rome  (18/9),  announced  the  discovery  of  a  bacillus  which 
they  supposed  to  be  the  cause  of  malarial  fevers— their  Bacillus  malariae. 


BACTERIA   IN   INFECTIOUS   DISEASES.  597 

The  writer  repeated  their  experiments  the  following  year  (1880)  in  the  vicin- 
ity of  New  Orleans,  and  reported  as  follows  : 

"Among1  the  organisms  found  upon  the  surface  of  swamp  mud  near 
New  Orleans,  and  in  the  gutters  within  the  city  limits,  are  some  which 
closely  resemble,  and  perhaps  are  identical  with,  the  Bacillus  malarias  of 
Klebs  and  Tommasi-Crudeli;  but  there  is  no  satisfactory  evidence  that  these 
or  any  of  the  other  bacterial  organisms  found  in  such  situations,  when  in- 
jected beneath  the  skin  of  a  rabbit,  give  rise  to  a  malarial  fever  corre- 
sponding with  the  ordinary  paludal  fevers  to  which  man  is  subject. 

"  The  evidence  upon  which  Klebs  and  Tommasi-Crudeli  have  based  their 
claim  of  the  discovery  of  a  Bacillus  malarise  cannot  be  accepted  as  sufficient; 
(a)  because  in  their  experiments  and  in  my  own  the  temperature  curve  in 
the  rabbits  experimented  upon  has  in  no  case  exhibited  a  marked  and  dis- 
tinctive paroxysmal  character ;  (6)  because  healthy  rabbits  sometimes  exhi- 
bit diurnal  variations  of  temperature  (resulting  apparently  from  changes  in 
the  external  temperature)  as  marked  as  those  shown  in  their  charts ;  (c)  be- 
cause changes  in  the  spleen  such  as  they  describe  are  not  evidence  of  death 
from  malarial  fever,  inasmuch  as  similar  changes  occur  in  the  spleens  of 
rabbits  dead  from  septicaemia  produced  by  the  subcutaneous  injection  of 
human  saliva;  (d)  because  the  presence  of  dark-colored  pigment  in  the 
spleen  of  a  rabbit  cannot  be  taken  as  evidence  of  death  from  malarial  fever, 
inasmuch  as  this  is  frequently  found  in  the  spleens  of  septicaemic  rabbits." 

Later  researches  have  also  failed  to  confirm  the  supposed  discovery  of 
Klebs  and  Tommasi-Crudeli,  and  it  is  now  generally  admitted  that  there  is 
no  satisfactory  evidence  in  favor  of  the  view  that  microorganisms  of  this 
class  are  concerned  in  the  etiology  of  the  malarial  fevers.  On  the  other 
hand,  we  have  now  very  extended  observations  which  indicate  that  the  blood 
parasite  discovered  by  Laveran  (1881)  in  the  blood  of  patients  suffering  from 
various  forms  of  malarial  fever  bears  an  etiological  relation  to  fevers  of  this 
class.  This  haematozodn  belongs  to  quite  a  different  class  of  microorgan- 
isms. It  was  first  described  by  Laveran  as  the  Oscillaria  malariee,  but  is 
more  frequently  spoken  of  at  present  as  the  Plasmodium  malarias. 

MALTA  FEVER. 

In  twelve  out  of  thirteen  cases  of  "Malta  fever  "Bruce  (1892)  found  a 
micrococcus  which  he  believes  to  be  the  cause  of  this  fever.  See  Micrococ- 
cus  of  Bruce  (No.  179). 

MALIGNANT   CEDEMA. 

See  Bacillus  oedematis  maligni  (No.  186). 

MASTITIS. 

In  ten  cases  of  puerperal  mastitis  Bumm  (1886)  found  Staphy- 
lococcus  pyogenes  aureus  in  seven  and  Streptococcus  pyogenes  in 
three.  In  a  case  reported  by  Sarpert  (1894)  diplococci  were  found 
corresponding  in  their  morphological  characters  with  the  gonococcus 
—the  patient  was  suffering  from  gonorrhoea. 

MASTITIS   IN   COWS. 

Bovine  mastitis  is  usually  due  to  infection  by  streptococci,  which  are  not 
always  the  same,  although  possibly  varieties  of  the  same  species.  See  Strep- 
tococcus of  Nocard  and  Mollereau  (No.  31),  Micrococcus  of  Kitt  (No.  21), 
Streptococcus  agalactia;  coiitagiosae  (No.  45).  Streptococcus  mastitis  spor- 
adise  (No.  45).  See  also  Bacilli  of  G-uillebeau  (No.  180). 


598  BACTERIA   IN  INFECTIOUS   DISEASES. 

Lucet  (1891)  in  twenty-two  cows  suffering  from  mastitis  obtained  in 
twelve  cases  a  motile  bacillus,  from  1  to  2  p  long,  which  did  not  liquefy 
gelatin  and  caused  a  development  of  gas  in  culture  media  (Bacillus  coli 
communis  ?). 

MASTITIS   IN   SHEEP. 
See  Micrococcus  of  gangrenous  mastitis  in  sheep  (No.  30). 

MEASLES. 

The  etiology  of  measles  and  of  the  specific  eruptive  febrile  diseases  gener- 
ally still  remains  unsettled.  The  occasional  presence  of  micrococci  in  the 
blood  of  patients  with  measles,  which  has  been  noted,  is  without  doubt  due 
to  a  secondary  or  mixed  infection  by  one  of  the  common  pyogenic  micro- 
cocci.  In  pneumonia  occurring  during  the  course  of  an  attack  of  measles 
the  Micrococcus  pneumonia?  crouposae  is  usually  found  in  the  pulmonary 
exudate. 

No  great  importance  can  be  attached  to  the  observations  made,  with  ref- 
erence to  the  presence  of  microorganisms  in  this  disease,  prior  to  the  intro- 
duction of  Koch's  plate  method  and  the  use  of  solid  culture  media  for  the 
differentiation  of  bacteria  similar  in  their  morphology.  In  1892  Canon  and 
Pielicke,  of  Berlin,  announced  the  discovery  of  a  minute  bacillus  in  the  blood 
of  patients  (fourteen)  with  measles,  but  their  discovery,  so  far  as  the  writer 
knows,  has  not  been  confirmed  by  more  recent  investigations.  See  Bacillus 
of  Canon  and  Pielicke  (No.  157). 

MENINGITIS. 
See  Cerebro-spinal  Meningitis. 

MICE,    INFECTIOUS    DISEASES  OF. 

See  Bacillus  typhi  murium  (No.  84)  and  Bacillus  of  Laser  (No.  83)  ;  also 
Bacillus  erysipelatos  suis  (No.  67),  which  appears  to  be  identical  with  Koch's 
Bacillus  of  septicaemia  in  mice ;  also  Bacillus  murisepticus  pleomorphus 
(No.  98). 

NEPHRITIS. 

The  various  microorganisms  which  have  occasionally  been  found  in  the 
urine  of  cases  of  chronic  nephritis  are  probably  not  directly  related  to  the 
renal  disease.  Numerous  observations  are  on  record  which  snow  that  patho- 
genic microorganisms  present  in  the  blood  or  tissues  may  find  their  way  into 
the  urine  during  the  course  of  the  acute  infectious  diseases.  In  these  cases 
it  is  probable  that  the  passage  of  bacteria  into  the  urine  depends  upon  struc- 
tural changes  in  the  kidneys,  due  to  the  presence  of  pathogenic  bacteria  or 
to  the  action  of  their  toxic  products.  Pernice  and  Scagliosi  (1894)  have  stud- 
ied the  development  of  nephritis  in  guinea-pigs,  dogs,  and  white  mice,  into 
which  they  injected  bouillon  cultures  of  various  pathogenic  bacteria — An- 
thrax bacillus,  Bacillus  pyocyaneus,  Staphylococcus  pyogenes  aureus,  Ba- 
cillus prodigiosus.  They  also  injected  filtered  cultures  of  these  bacilli.  As 
a  result  of  their  experiments  they  conclude  that  the  appearance  of  microor- 
ganisms in  the  urine  in  acute  infectious  diseases  depends  upon  pathological 
anatomical  changes  in  the  kidneys,  which  may  result  either  from  the  jm  s 
ence  of  the  bacteria  or  from  the  action  of  their  toxic  products.  Pathogenic 
bacteria  are  not  infrequently  found  in  the  urine  in  the  acute  infectious  dis- 
eases of  man — e.g.,  typhoid  fever,  pneumonia,  septicaemia ;  and  in  certain 
cases  of  mixed  infection  bacteria  may  be  found  in  the  urine  which  have  no 


BACTERIA   IN   INFECTIOUS   DISEASES.  599 

direct  etiological  relation  to  the  specific  infectious  disease  from  which  the  pa- 
tient is  suffering — e.gr.,  staphylococci  in  cases  of  measles,  or  streptococci  in 
cases  of  diphtheria. 

Ascending  nephritis  is  an  infectious  process,  usually  due  to  Bacillus  coli 
communis  (See  pyelonephritis). 

Letzerich  (1887)  has  described  a  form  of  nephritis  which  he  ascribes  to  a 
bacillus  found  by  him  in  the  urine  and  in  sections  of  the  kidneys  of  rabbits 
inoculated  with  a  culture  of  this  bacillus. 

Lustgarten  arid  Manneberg  (1887)  in  three  cases  of  acute  Bright's  disease 
found  streptococci  in  the  urine,  which  they  suppose  to  have  had  an  etiologi- 
cal  relation  to  the  renal  disease.  The  following  year  Manneberg  reported 
eleven  additional  cases,  in  eight  of  which  he  found  the  same  streptococcus,- 
which  he  believes  to  be  different  from  Streptococcus  pyogenes,  but  this  can- 
not be  considered  as  established.  Nor  has  he  shown  that  the  streptococcus 
obtained  by  him  from  the  urine  was  present  in  the  kidneys  of  his  patients, 
or  that  pure  cultures  of  this  streptococcus  produce  acute  nephritis  when  in- 
oculated into  lower  animals. 


OPHTHALMIA. 

Although  various  pathogenic  bacteria  are  frequently  found  in 
healthy  eyes,  there  can  be  no  doubt  that  acute  and  chronic  inflamma- 
tions here,  as  elsewhere,  are  commonly  due  to  the  presence  of  micro- 
organisms. In  gonorrhoeal  ophthalmia  the  "  gonococcus  "  of  Neisser 
is  the  infectious  agent.  According  to  Fuchs  (1894)  a  considerable 
proportion  of  the  cases  of  so-called  Egyptian  ophthalmia  are  due  to 
infection  by  the  gonococcus,  while  in  another  group  of  cases  the  in- 
fectious agent  is  the  bacillus  of  Koch  and  Kartulis  (No.  138).  Cer- 
tain cases  are  also  due  to  a  mixed  infection  resulting  from  the  pres- 
ence of  both  of  these  pathogenic  microorganisms.  Demetriades  (1894) 
says  that  the  gonococcus  found  in  cases  of  Egyptian  ophthalmia  is 
much  smaller  than  that  encountered  in  cases  of  gonorrhoea ;  but  that 
it  is  the  same  was  demonstrated  by  Kartulis,  who  introduced  pus 
from  the  eye  of  a  patient,  containing  this  coccus,  into  the  urethra  of 
a  native,  who  developed  a  typical  gonorrhoea  at  the  end  of  twenty- 
four  hours  as  a  result  of  the  inoculation. 

Perles  (1895)  has  made  numerous  inoculation  experiments  in  the 
eyes  of  rabbits  and  reports  the  following  results :  Pure  cultures  of 
Bacillus  subtilis,  of  the  cholera  spirillum,  and  of  various  non-patho- 
genic saprophytes,  introduced  into  the  anterior  chamber  or  the  vit- 
reous, caused  no  perceptible  changes.  Typhoid  bacilli  introduced 
into  the  anterior  chamber  caused  hypopyon  and  in  the  vitreous  an 
abscess.  Streptococci  gave  rise  to  an  exudate  in  the  anterior  cham- 
ber and  to  pus  formation  in  the  vitreous.  Diphtheria  bacilli  caused 
a  purulent  exudate  in  the  anterior  chamber  with  a  moderate  kerato- 
iritis,  and  abscess  formation  when  introduced  into  the  vitreous. 
Friedlander's  bacillus,  in  the  vitreous,  caused  a  severe  panophthal- 
mitis,  which  led  to  rupture  of  the  eyeball  at  the  end  of  sixteen  hours ; 


600  BACTERIA  IN   INFECTIOUS   DISEASES. 

in  the  anterior*  chamber  the  result  was  similar,  but  not  so  rapidly 
induced.  No  infection  occurs  through  the  uninjured  conjunctiva. 
When  the  pneumonia  coccus  was  introduced  into  the  eye  of  a  rabbit 
general  infection  and  death  from  septica3mia  quickly  followed.  Ac- 
cording to  Gasparrini  the  micrococcus  of  pneumonia  is  found  in  the 
conjunctival  sac  in  a  large  proportion  of  healthy  eyes — in  8  out  of  lo 
of  the  100  students  examined  by  him.  When  injected  into  the  vit- 
reous or  anterior  chamber,  in  rabbits,  fresh  cultures  gave  rise  to  a 
.panophthalmia,  and  cultures  four  or  five  days  old  to  a  plastic  iritis 
or  to  atrophy  of  the  eye  from  a  chronic  infectious  process.  In  cases 
of  kerato-hypopyon  in  man  (21)  and  of  panophthalmia  (4)  this  mi- 
crococcus was  found,  and  in  six  of  the  first-mentioned  cases  it  was  so 
virulent  that  it  killed  rabbits,  when  injected  subcutaneously,  in  from 
twenty-two  to  thirty-six  hours.  In  seven  other  cases  it  was  found  in 
pure  culture,  but  proved  not  to  be  virulent — i.e.,  did  not  kill  rabbits. 
In  eight  cases  it  was  associated  with  staphylococci.  Sattler,  in  a  case 
of  panophthalmia  resulting  from  injury  by  a  splinter  of  wood,  ob- 
tained cultures  of  Bacillus  pyocyaneus;  Randolph  reports  a  case 
caused  by  Bacillus  coli  communis;  Wagenmann  states  that  in  most 
cases  in  which  purulent  infiltration  of  the  vitreous  follows  a  perfora- 
ting wound  of  the  eye  the  ordinary  pus  cocci  are  found.  In  the  me- 
tastatic  eye  affections  occurring  in  the  course  of  puerperal  septi- 
ca3mia,  Herrnheiser  (1892)  obtained  in  two  cases  (of  retinitis  septica) 
very  virulent  cultures  of  Streptococcus  pyogenes  and  in  one  a  culture 
of  Staphlyococcus  pyogenes  aureus.  In  a  case  of  metastatic  pan- 
ophthalmia,  occurring  in  a  man  aged  sixty-seven,  after  an  attack 
of  pneumonia,  the  micrococcus  of  pneumonia  was  found.  Accord- 
ing to  Axenfeld  (1894)  the  last-mentioned  microorganism  is  a  fre- 
quent cause  of  purulent  metastatic  ophthalmia. 

OSTEOMYELITIS  AND   PERIOSTITIS. 

The  evidence  with  reference  to  the  presence  of  Staphylococcus 
pyogenes  aureus  in  acute  osteomyelitis  and  its  probable  etiological 
relation  to  the  cases  in  which  it  is  found,  is  given  in  the  article  de- 
scriptive of  this  microorganism;  but  the  researches  of  Kraske  (lSs»>) 
and  of  Lannelongue  and  Achard  (1890)  show  that  the  "golden  sta- 
phylococcus  "  is  not  always  found  in  osteomyelitis.  The  last-named 
investigators,  in  a  series  of  thirteen  cases,  found  Staphylococcus  py<  >- 
genes  aureus  in  four  only,  and  in  one  of  these  Staphylococcus  pyo- 
genes albus  was  also  present;  in  three  cases  Staphylococcus  pyogem  s 
albus  was  obtained  in  pure  cultures;  in  two  cases  it  was  associated 
with  Streptococcus  pyogenes;  and  in  two  cases  a  streptococcus  ^  as 
found  which  resembled  Streptococcus  pyogenes  and  yet  differed  from 


BACTERIA   IN   INFECTIOUS   DISEASES.  601 

it  in  some  particulars.  The  same  bacteriologists  found  the  pneu- 
monia coccus  in  two  cases  in  children.  Fisher  and  Levy  (1893)  also 
report  two  cases,  in  children,  in  which  this  micrococcus  was  found 
— one  a  fatal  case  of  meningitis.  In  two  other  cases  streptococcus 
pyogenes  was  obtained  in  pure  cultures.  The  typhoid  bacillus  has 
also  been  found  by  several  investigators — Ebermayer,  Orion0,  Colzi, 
Ullmann.  It  is  therefore  evident  that  osteomyelitis  cannot  be  con- 
sidered a  specific  affection;  on  the  other  hand,  as  in  abscesses  de- 
veloped in  the  cellular  tissue,  in  glands,  or  in  the  various  organs,  it 
is  to  be  regarded  as  a  localized  infectious  process  which  may  be 
induced  by  various  pathogenic  microorganisms  which  through  some 
channel  have  effected  a  lodgment  in  the  blood  or  tissues  of  the  body. 
The  exciting  cause  of  a  peri  osteal  inflammation  is,  no  doubt,  not  in- 
frequently an  injury  of  some  kind.  Chronic  periostitis  and  osteo- 
myelitis are  developed  in  a  similar  way  as  a  result  of  a  localized 
tubercular  infection. 

OTITIS  MEDIA. 

In  otitis  media  various  microorganisms  have  been  found  in  pus 
obtained  by  paracentesis  of  the  tympanum,  as  well  as  in  the  chronic 
discharge  after  perforation ;  and  there  can  be  but  little  doubt  that 
these  microorganisms  are  responsible,  directly  or  indirectly,  for  the 
inflammatory  process  and  pus  formation.  The  following  species  are 
most  frequently  found  in  the  purulent  discharge  in  recent  cases  of 
otitis  media :  Micrococcus  pneumonia  crouposa3  ("  Diplococcus  pneu- 
moniaa  "),  Streptococcus  pyogenes,  Staphylococcus  pyogenes  albus, 
Staphylococcus  pyogenes  aureus,  Friedlander's  bacillus.  The  fol- 
lowing have  also  been  found  occasionally:  Staphylococcus  tenuis, 
Bacillus  tenuis,  Micrococcus  tetragenus,  Bacillus  pyocyaneus. 

According  to  Zaufal,  Micrococcus  pneumonia  crouposa3  is  most 
frequently  found  in  cases  which  result  from  exposure  to  cold,  while 
the  ordinary  pus  cocci  are  more  frequently  found  in  otitis  which  is 
secondary  to  specific  febrile  diseases. 

Martha  (1892)  reports  two  cases  in  which  Bacillus  pyocyaneus 
was  present  in  pure  culture — in  fifty-one  other  cases  examined  this 
bacillus  was  not  found.  This  bacillus  has  also  been  found  occasion- 
ally by  other  investigators— Pes  and  Gradenigo  (1894),  Hartmann 
(1894),  Kossel  (1894). 

Scheibe  (1892)  in  sixteen  cases  of  mastoid  abscess  following  mid- 
dle-ear disease  found  the  micrococcus  of  pneumonia  in  six,  Strepto- 
coccus pyogenes  in  five,  Staphylococci  in  one,  and  an  undetermined 
micrococcus  in  one. 

Stern  (1895)  in  thirty  cases  of  chronic  purulent  otitis  media  made 
bacteriological  examinations  with  the  following  result :  Staphylococ- 
42 


602  BACTERIA   IN   INFECTIOUS   DISEASES. 

cus  pyogenes  albus  was  found  in  six,  Staphylococcus  pyogenes  aureus 
in  two,  Streptococcus  pyogenes  in  three,  Bacillus  coli  communis  in 
one,  and  various  bacilli,  vibrios,  and  cocci  in  the  remaining  cases; 
some  of  these  were  fluorescent,  some  produced  a  foetid  odor,  etc. 


OZ^ENA. 

The  researches  of  Thost,  Klamann,  Hajek,  and  others  show  that 
Friedlander's  bacillus  is  present  in  the  nasal  secretions  in  a  consider- 
able proportion  of  the  cases  of  ozsena,  but  its  etiological  relation  to 
the  morbid  condition  which  gives  rise  to  the  offensive  discharge  has 
not  been  established. 

Thost  found  this  bacillus  in  twelve  out  of  seventeen  cases  studied 
by  him,  and  frequently  almost  in  a  pure  culture;  but  he  also  found 
it  in  rhinitis  from  syphilitic  ulceration,  from  polypus,  and  in  simple 
coryza. 

Hajek  found  Friedlander's  bacillus  in  seven  out  of  ten  cases  stud- 
ied by  him,  but  it  was  associated  with  various  other  species  of  bac- 
teria, and  especially  with  the  pyogenic  micrococci  and  with  Bacillus 
fluorescens  liquefaciens.  He  also  obtained  almost  constantly  his 
Bacillus  fcetidus  ozsense  (No.  Ill),  which  appears  to  have  been  the 
cause  of  the  foetid  odor  of  the  nasal  discharge. 

Marsano  (1890)  in  ten  cases  of  ozsena  found  a  capsule  bacillus  in 
the  nasal  secretions  which  closely  resembles  Friedlander's  bacillus, 
but  which  he  believes  not  to  be  identical  with  it. 

Abel  (1893)  in  sixteen  cases  of  "  oza3na  simplex  "  found  a  similar 
capsule  bacillus,  but  he  arrives  at  the  conclusion  that  it  is  not  iden- 
tical with  Friedlander's  bacillus,  and  believes  it  to  be  the  specific 
cause  of  rhinitis  atrophicans  fcetida.  According  to  Abel  his  bacillus 
resembles  Pfeiffer's  capsule  bacillus^  (No.  80)  more  closely  than  it 
does  that  of  Friedlander,  and  it  is  almost  identical  with  the  capsule 
bacillus  of  Fasching  (No.  150).  This  bacillus  is  described  by  Abel 
under  the  name  of  Bacillus  mucosus  Ozsena?.  It  is  said  to  be  differ- 
entiated from  the  bacillus  of  rhinoscleroma  and  Bacillus  sputigenus 
crassus  of  Kreibohm,  by  the  fact  that  it  does  not  stain  by  Gram's 
method. 

Strazza  (1893)  in  twenty-five  cases  examined  found  a  capsule 
bacillus  constantly  associated  with  streptococci  and  staphylococci. 
This  bacillus  was  not  found  in  cases  of  rhinitis  chronica  simplex  or 
of  rhinitis  syphilitica.  According  to  Strazza,  also,  this  bacillus  is 
differentiated  from  the  bacillus  of  rhinoscleroma  bjr  the  fact  that  it 
does  not  stain  by  Gram's  method ;  it  is  said  to  resemble  Pfeiffer's 
bacillus  in  cultures,  but  to  be  somewhat  smaller. 

Loewenberg  (1894)  has  called  attention  to   the  fact  that  he  re- 


BACTERIA   IN   INFECTIOUS   DISEASES.  603 

ported  in  1881  that  he  had  always  found  in  oza3na  a  microbe  sur- 
rounded by  a  capsule  and  not  staining  by  Gram's  method.  He  now 
says  that  this  microbe  corresponds  with  Friedlander's  bacillus  in 
form  and  staining  reactions,  but  differs  from  it  in  other  particulars 
as  follows :  It  shows  a  very  scanty  development  in  milk  and  causes 
no  perceptible  change  in  this  medium.  Friedlander's  bacillus,  on  the 
other  hand,  coagulates  milk  and  its  growth  is  often  attended  with  an 
evolution  of  gas.  The  two  bacilli  also  give  rise  to  different  odors. 
The  bacillus  of  Friedlander  gives  off  from  gelatin  and  bouillon  cul- 
tures an  odor  of  trimethylamine  and  old  gelatin  plates  give  off  the 
odor  of  old  cheese.  Cultures  of  the  bacillus  of  ozaBna,  on  the  con- 
trary, give  off  an  agreeable  odor.  The  offensive  odor  characteristic 
of  oza3na  is  never  given  off  from  cultures  of  this  bacillus.  Loewen- 
berg  concludes  from  his  investigations  that  the  microbe  of  ozsena  is 
specifically  distinct  from  the  bacillus  of  Friendlander  and  that  it 
bears  an  etiological  relation  to  this  disease. 

PANARITIUM. 

According-  to  Saint-Sevrin  (1894)  panaritium  (Panaris  des  pecheurs)  is 
very  common  among  the  fisherman  of  the  island  of  Newfoundland  and  of 
the  North  Sea.  His  researches  lead  him  to  conclude  that  it  is  due  to  infection 
by  a  micrococcus  which  produces  a  red  pigment  (microbe  rouge  de  la  sardine) 
in  association  with  an  anaerobic  bacillus.  The  coccus  is  from  0.5  to  0.6  /*  in 
diameter  and  is  usually  seen  in  pairs ;  it  liquefies  gelatin  and  produces  a 
carmine-red  pigment. 

It  is  probable  that  the  common  pus  cocci  are  usually  concerned  in  the 
etiology  of  "felons."  In  a  case  reported  by  Huber  Staphylococcus  pyogenes 
albus  was  obtained  in  pure  culture  from  the  pus  of  a  panaritium  and  also  in 
blood  obtained  from  a  finger  of  the  opposite  hand.  Bernheim  obtained  the 
colon  bacillus  from  the  pus  of  a  panaritium  developed  in  the  course  of  an 
attack  of  typhoid  fever. 

PAROTITIS. 

No  demonstration  of  a  specific  microorganism  in  mumps  has  been  made, 
but  in  non-specific,  suppurative  parotitis  one  or  other  of  the  pyogenic  micro- 
cocci  appears  to  be  the  cause  of  the  inflammation  and  pus  formation.  In 
parotitis  occurring  as  a  complication  of  pneumonia  Micrococcus  pneumonias 
crouposse  has  been  found  as  the  only  microorganism  in  pus  from  the  inflamed 
gland  (Testi,  Duplay).  Letzerich  in  1895  made  a  preliminary  communication 
in  which  he  claimed  to  have  discovered  microorganisms  in  the  blood  of 
patients  with  mumps.  These  he  describes  as  "large,  round  spores;"  no 
bacilli  were  found. 

In  a  fatal  case  of  typhoid  fever  in  which  a  suppurative  inflammation  of 
the  parotid  gland  was  found,  Janowski  (1895)  obtained  a  pure  culture  of  Ba- 
cillus typhi  abdominalis  from  the  pus  of  the  parotid  abscess. 

PEMPHIGUS. 

Demme  (1886)  has  cultivated  a  diplococcus  from  a  case  of  acute  pemphi- 
gus which  possibly  is  related  to  this  disease  (see  Micrococcus  of  Demme,  No. 
27,  page  331).  The  same  coccus  was  found  by  Dahnhardt  in  a  similar  case. 


604  BACTERIA  IN  INFECTIOUS  DISEASES. 

Strelitz  (1892)  obtained  in  cultures  from  pemphigus  vesicles  a  micrococcus 
which  corresponded  in  every  respect  with  Staphylococcus  pyogenes  aureus. 
Inoculation  of  this  micrococcus  in  his  own  arm  caused  the  development  of 
typical  pemphigus  bullae. 

PERICARDITIS. 

Pericarditis  is  a  localized  infectious  process  due  to  various  patho- 
genic microorganisms.  In  two  cases  reported  by  Barbacci  (1892)  the 
micrococcus  of  pneumonia  was  found  to  be  the  infectious  agent. 
Paviot  (1894)  reports  a  fatal  case  of  purulent  pericarditis  in  which  a 
diplococcus  was  found  resembling  Friedlander's  bacillus.  Ernst 
(1893)  obtained  from  the  pericardial  sac,  in  a  case  in  which  the  tu- 
bercle bacillus  was  also  present,  a  variety  of  Bacillus  pyocyaneus. 

In  "  uraemic  pericarditis  "  Banti  failed,  in  four  cases,  to  find  any 
microorganism  in  fluid  obtained  from  the  pericardial  sac. 

In  pericarditis  occurring  in  general  septica3mic  infection  the 
microorganism  to  which  the  general  infection  is  due  will  probably 
be  found  in  the  pericardial  sac,  and  when  it  occurs  as  a  complication 
of  one  of  the  specific  infectious  diseases  in  which  bacteria  are  usually 
not  found  in  the  blood — eruptive  fevers — it  is  probably  due  to  a  mixed 
infection  with  one  of  the  common  pus  cocci.  In  chronic  tubercular 
pericarditis  the  tubercle  bacillus  is  the  infectious  agent. 

PERITONITIS. 

That  peritonitis  usually  results  from  the  presence  of  microorgan- 
isms in  the  cavity  of  the  abdomen  seems  to  be  well  established  by  ex- 
perimental evidence  and  by  bacteriological  researches  in  cases  of  this 
disease.  Mechanical  irritants,  like  finely  powdered  glass  (writer's 
experiments) ,  introduced  into  the  cavity  of  the  abdomen  of  rabbits, 
do  not  cause  peritonitis  unless  microorganisms  are  introduced  at  the 
same  time ;  the  minute  fragments  of  glass  become  encysted  and  the 
animal  remains  in  good  health.  But  Pernice  has  shown  that  peri- 
tonitis may  be  induced  in  rabbits  and  in  guinea-pigs  by  injecting 
into  the  cavity  of  the  abdomen  various  chemical  substances,  such  as 
concentrated  mineral  acids,  acetic  acid,  phenol,  nitrate  of  silver,  etc. 
It  is  also  demonstrated  by  numerous  experiments  that  pure  cultures 
of  various  bacteria  injected  into  the  cavity  of  the  abdomen  of  the 
animals  mentioned  may  produce  a  fibrinous  or  a  purulent  peritonitis. 
Among  these  is  the  Bacillus  coli  communis,  which  is  constantly 
present  in  the  intestine  of  healthy  persons;  and  in  peritonitis  follow- 
ing perforation  of  the  bowels  this  bacillus  is  responsible,  in  part  at 
least,  for  the  intense  peritoneal  inflammation  which  so  quickly  occurs. 
In  puerperal  peritonitis  the  pus  cocci,  and  especially  Streptococcus 
pyogenes,  appear  to  be  the  usual  cause  of  the  inflammatory  process. 


BACTERIA   IN   INFECTIOUS   DISEASES.  605 

Weichselbaum  has  observed  two  cases  of  primary  peritonitis  and 
pleuritis  apparently  induced  by  Micrococcus  pneumonias  crou  posse, 
as  this  microorganism  was  found  in  the  exudate  into  the  peritoneal 
cavity.  The  same  author,  in  a  case  of  peritonitis  resulting  from 
rupture  of  the  spleen  in  the  course  of  typhoid  fever,  obtained  a  pure 
culture  of  the  typhoid  bacillus  from  the  peritoneal  cavity.  The  re- 
sults of  A.  FrankePs  researches  (1891)  are  as  follows :  In  thirty-one 
cases  examined  pure  cultures  were  obtained  in  twenty,  viz. :  Bacil- 
lus coli  communis,  nine  times;  streptococci,  seven  times;  Bacillus 
lactis  aerogenes,  twice;  Micrococcus  pneumonia  crouposas,  once; 
Staphylococcus  pyogenes  aureus,  once.  In  three  cases  Bacillus  coli 
communis  was  present  in  association  with  other  bacilli,  and  in  four 
cases  the  bacteriological  examination  gave  a  negative  result. 

Frankel  has  also  shown  that  pure  cultures  of  Bacillus  coli  com- 
munis injected  into  the  cavity  of  the  abdomen  of  rabbits  cause  a 
typical  peritonitis.  The  present  writer  has  frequently  obtained  the 
same  result  in  experiments  made  with  this  bacillus.  It  would  ap- 
pear, therefore,  that  the  peritonitis  which  so  constantly  results  from 
wounds  of  the  intestine  is  probably  due,  to  a  considerable  extent,  to 
the  introduction  of  this  microorganism  from  the  lumen  of  the  intes- 
tine, where  it  is  constantly  found,  into  the  peritoneal  cavity,  where 
the  conditions  are  favorable  for  its  rapid  development. 

Malvoz  in  1893  found  Bacillus  coli  communis,  for  the  most  part 
in  pure  cultures,  in  five  out  of  seven  cases  examined  by  him ;  in  the 
other  two  cases  he  found  Streptococcus  pyogenes  in  one  and  a  bacil- 
lus which  appeared  to  be  identical  with  Bacillus  typhi  abdominalis 
in  one.  Barbacci  (1892)  in  two  cases  in  which  meningitis  was  also 
present  (in  one  endocarditis  also)  found  the  micrococcus  of  pneu- 
monia in  pure  cultures.  Le  Gendre  (1895)  has  reported  a  case  in 
which  the  same  microorganism  was  alone  present,  and  states  that 
in  a  search  of  the  literature  he  finds  eleven  recorded  cases  due  to  this 
micrococcus;  of  these  eight  terminated  fatally.  Tavel  and  Lanz 
(1893)  in  a  series  of  seventy-two  cases  examined  found  bacteria  re- 
sembling the  colon  bacillus  in  thirty-one.  Flexner  (1893)  reports  a 
case  of  peritonitis  caused  by  Proteus  vulgaris.  Tubercular  peri- 
tonitis is,  of  course,  due  to  infection  by  the  tubercle  bacillus. 

PLANTS,    INFECTIOUS   DISEASES   OF. 

The  infectious  diseases  of  plants  are,  for  the  most  part,  due  to  parasitic 
fungi,  but  several  infectious  plant  diseases  have  been  shown  to  depend  upon 
the  presence  of  bacteria  in  the  diseased  tissues,  and  in  others  this  has  been 
claimed  by  investigators  upon  more  or  less  satisfactory  evidence.  The  lim- 
its of  the  present  volume  only  admit  of  an  enumeration  of  the  most  impor- 
tant of  these  bacteria : 

Micrococcus  amylovorus  (Burrill)  is  believed  to  be  the  cause  of  "pear 
blight;"  Bacillus  sorghi  (Kellerman  and  Swingle)  of  "sorghum  blight;'* 


606  BACTERIA   IN   INFECTIOUS   DISEASES. 

Bacillus  hyacinth!  septicus  (Heinz)  of  an  infectious  disease  of  hyacinths  ;  Ba- 
cillus amylobacter  of  potato  rot  (Nassfaule) ;  Bacillus  tracheiphilus  (E.  F. 
Smith)  of  blight  in  melons  and  other  cucurbitacese. 

PLEURITIS, 

The  usual  infectious  agent  in  acute  fibrinous  pleurisy  accompany- 
ing pneumonia  is  Micrococcus  pneumonia?  crouposa?  (No.  8).  Net- 
ter  (1892)  reports  that  in  66  cases  of  genuine  fibrinous  pleurisy  in 
which  he  has  made  bacteriological  researches  since  1886,  he  has 
always  found  this  micrococcus.  In  cases  in  which  culture  experi- 
ments give  a  negative  result,  this  is,  according  to  Netter,  due  to  the 
fact  that  the  micrococci  are  apt  to  perish  at  the  crisis  of  the  disease. 
These  cases  do  not  usually  result  in  empyema  and  run  a  more  favor- 
able course  than  those  in  which  the  pus  cocci  are  present.  Prudden 
(1893)  in  21  cases  of  sero-fibrinous  pleurisy  failed  to  find  any  bac- 
teria in  the  exudate  in  1*2,  and  found  the  micrococcus  of  pneumonia 
in  2  only.  Lemoine  (1895)  also  reports  that  in  28  cases,  out  of  32 
examined  by  him,  the  exudate  was  entirely  sterile;  in  4  cases  he 
found  Staphylococcus  pyogenes  albus.  The  remaining  cases  were  of 
tubercular  origin. 

Levy  (1895),  in  reviewing  the  literature  of  the  subject,  arrives  at 
the  conclusion  that  the  micrococcus  of  pneumonia  is  the  usual  cause 
of  pleurisy  in  children  and  of  metapneumonic  pleurisy,  but  that  in 
metastatic,  pysemic,  pleuritic  inflammation  streptococci  or  staphylo- 
cocci  are  the  usual  infectious  agents.  Pleurisy  due  to  streptococcus 
or  staphlyococcus  infection  is  not  in  all  cases  attended  with  pus  for- 
mation ;  the  exudate,  in  a  certain  proportion  of  the  cases,  may  remain 
serous  (Levy,  Ludwig,  Goldschneider) . 

The  micrococcus  of  pneumonia  was  found  by  Jakowski  (1892)  in 
21  out  of  34  cases  in  which  pure  cultures  were  obtained ;  of  the  re- 
maining cases  streptococci  were  found  in  10,  Staphylococcus  pyogenes 
aureus  in  1,  and  the  tubercle  bacillus  in  2.  In  14  cases  of  mixed 
infection  Staphylococcus  pyogenes  aureus  and  albus  were  found  in 
6,  Friedlander's  bacillus  and  streptococci  in  1;  the  micrococcus  of 
pneumonia  with  streptococci  in  1,  with  Staphylococcus  pyogenes 
albus  in  2,  and  with  Staphylococcus  pyogenes  aureus  in  1.  In  7 
cases  no  bacteria  were  found.  According  to  Jakowski  those  cases 
in  which  no  bacteria  are  obtained  in  cultures  are  usually  due  to 
tubercular  infection.  Goldschneider  (1892)  reports  4  cases  of  serous 
pleurisy,  in  3  of  which  he  found  Streptococcus  pyogenes  and  in  1 
Staphylococcus  pyogenes  aureus.  Bordoni-Uffreduzzi  (1895)  has  re- 
ported a  case  of  double  pleurisy  in  a  girl,  aged  twelve,  who  was 
assaulted  by  an  individual  with  gonorrhoea,  in  which  the  gonococ- 
cus  was  the  only  microorganism  present  in  the  pleural  exudate. 


BACTERIA   IN   INFECTIOUS   DISEASES.  60? 

In  pleurisy  occurring  as  a  complication  of  typhoid  fever  the  ty- 
phoid bacillus  has  been  found  in  the  exudate  (sometimes  serous  and 
sometimes  purulent)  by  several  bacteriologists.  Bacillus  coli  com- 
munis  has  also  been  found  (Albarran  and  Halle).  According  to  the 
statistics  of  Flemming  about  41  per  cent  of  the  fatal  cases  of  pleurisy 
(424  cases  examined)  are  due  to  tubercular  infection. 

PLEURO-PNEUMONIA   OF  CATTLE. 

The  evidence  appears  to  be  satisfactory  that  this  infectious  disease 
is  due  to  the  Pneumobacillus  liquefaciens  bovis  of  Arloing  (No.  120). 

PLEURO-PNEUMONIA    (SEPTIC)   OF   CALVES. 

An  infectious  disease  of  calves,  described  by  Galtier  as  a  septic  pleuro- 
pneumonia,  or  pneumo-enteritis,  is  apparently  due  to  the  Pneumobacillus 
septicus  (No.  182)  of  the  author  named. 

PNEUMONIA. 

The  usual  infectious  agent  in  croupous  pneumonia  is  Micrococcus 
pneumonise  crouposa3  (No.  8).  Friedlander's  bacillus  and  other  mi- 
croorganisms have  been  found  in  a  comparatively  small  proportion 
of  the  cases ;  but  it  is  probable  that  some  of  these  at  least  were  due 
to  a  mixed  infection  and  that  the  specific  infectious  agent  was  over- 
looked. (See  also  Broncho-pneumonia.) 

PNEUMONIA  IN    HORSES. 
See  Diplococcus  of  pneumonia  in  horses,  of  Schutz,  No.  32. 

PNEUMO-ENTERITIS   OF   SWINE. 
See  Swine  plague. 

PSEUDO-LEUKEMIA. 

Various  microorganisms  have  been  found  in  connection  with  pseudo-leu- 
kaemia, but  no  one  of  these  has  been  shown  to  bear  a  specific  etiological  re- 
lation to  the  disease.  In  certain  cases  diagnosed  as  pseudo-leukaemia  tuber- 
cular infection  of  the  lymphatic  glands  and  spleen  has  been  found  at  the 
autopsy  (Weishaupt).  In  a  case  reported  by  Verdelli  (1891)  the  ordinary 
pyogenic  micrococci  were  obtained  from  the  blood  and  lymphatic  glands. 
Gabbi  and  Barbacci  (1892)  report  a  case  in  which  a  virulent  variety  of  the 
colon  bacillus  was  obtained  from  blood  drawn  from  a  finger,  and  from  the 
spleen  and  lymphatic  glands  after  death.  In  another  case  no  microorgan- 
isms could  be  found.  Traversa  (1893)  obtained  a  streptococcus  (probably 
Streptococcus  pyogenes)  in  pure  cultures  from  the  blood,  in  a  case  which 
came  under  his  observation.  Grossi  (1893),  in  a  case  in  which  he  had  an 
opportunity  to  make  a  post-mortem  examination,  failed  to  find  any  microor- 
ganisms in  the  blood,  the  lymphatic  glands,  or  in  serum  from  the  cedematous 
tissues. 


608  BACTERIA  IN  INFECTIOUS   DISEASES. 

PSEUDO-TUBERCULOSIS. 

Preisz  (1894)  has  compared  the  bacillus  of  pseudo-tuberculosis  described 
by  Npcard  with  that  of  Pfeiffer,  of  Parietti,  and  of  Zagari,  and  finds  them 
identical  (see  Bacillus  pseudo-tuberculosis,  No.  121).  A  different  bacillus  of 
pseudo-tuberculosis  was  obtained  by  Preisz  from  an  infected  sheep  (No.  183). 
Kutscher  (1894)  has  described  a  bacillus  which  produces  a  pseudo-tubercu- 
losis in  mice,  under  the  name  Bacillus  pseudo-tubercularis  murium  (No.  184). 
Eberth  (1886)  has  described  a  bacillus  of  pseudo-tuberculosis  in  rabbits  which 
appears  to  be  identical  with  the  microorganism  of  **  zooglcea-tuberculosis  "  of 
Malasses,  Vignal,  and  Chantemesse. 


PUERPERAL   FEVER. 

Puerperal  fever  is  usually  due  to  infection  by  Streptococcus  pyo- 
genes  (No.  5),  and  in  fatal  cases  this  microorganism  is  almost  always 
found.  In  a  few  fatal  cases  Staphylococcus  aureus  has  been  found 
in  pure  cultures  (Brieger,  Fehling,  Doderlein,  and  others),  and  in 
non-fatal  cases  of  a  comparatively  mild  character  staphylococci  are 
not  infrequently  the  cause  of  the  infection  and  accompanying  fever. 
Doderlein  has  reported  an  epidemic,  of  limited  extent,  in  which  there 
was  a  mixed  infection  by  streptococci  and  staphylococci.  Sanger, 
Kronig  (1893),  and  others  have  reported  cases  in  which  fever,  devel- 
oped during  the  puerperium,  was  apparently  due  to  the  presence  of 
gonococci  in  the  uterus. 

Von  Frangue  (1893)  and  Eisenhart  (1884)  have  reported  cases  in 
which  Bacillus  coli  communis  was  the  infectious  agent. 

PURPURA   H^EMORRHAGICA. 

See  account  of  bacilli  found  in  purpura  ha3morrhagica  by  Babes 
(No.  146),  Kolb  (No.  147),  and  Tizzoni  and  Giovannini  (No.  145). 

PYAEMIA. 

Pathologists  at  the  present  day  include  all  cases  of  general  blood  infection 
under  the  name  septicaemia,  and  localized  infections  have  special  names — 
e.g.,  mastitis,  pleuritis,  peritonitis,  metastatic  abscess,  etc.  When  the  toxic 
products  of  pathogenic  bacteria  are  absorbed  from  the  surface  of  a  suppurat- 
ing wound,  from  an  abscess  cavity,  or  from  any  localized  focus  of  infection, 
we  have  a  septic  toxaemia,  which  manifests  itself  by  more  or  less  fever,  and 
by  various  symptoms  connected  with  the  nervous  system,  etc.  The  use  of 
these  terms  in  the  sense  indicated  seems  to  do  away  with  the  necessity  for 
using  the  old  term  pyaemia. 

PYELONEPHRITIS. 

In  ascending  nephritis  or  pyelonephritis,  which  is  very  commonly 
secondary  to  a  cystitis  of  long  standing,  there  is  good  reason  to  be- 
lieve that  the  inflammatory  changes  and  pus  formation  depend  upon 
the  presence  of  certain  bacteria  which  are  found  in  the  urine  of  such 


BACTERIA   IN   INFECTIOUS   DISEASES.  609 

patients  during  life  and  in  the  diseased  kidney  removed  by  surgical 
operation  or  post-mortem.  And  recent  researches  show  that  the  Ba- 
cillus coli  communis,  which  is  constantly  present  in  the  intestine  of 
healthy  individuals,  is  found  more  frequently  than  any  other  micro- 
organism in  the  so-called  "surgical  kidney." 

The  most  important  and  comprehensive  work  upon  the  bacteriology  of 
pyelonephritis  is  that  of  Schmidt  and  Aschoff,  published  in  Jena  in  1893. 
The  authors  named  give  a  complete  resume  of  the  literature  of  the  subject, 
and  a  full  report  of  fourteen  cases  of  pyelonephritis,  in  which  they  have 
made  bacteriological  investigations.  They  also  report  a  series  of  experiments 
upon  rabbits,  in  which  injections  of  a  pure  culture  of  the  Bacillus  coli  com- 
munis were  made  into  the  left  ureter,  after  tying  it  below  the  point  of  in- 
jection. The  ligature  was  removed  after  the  injection  had  been  made,  and 
the  wound  in  the  abdominal  wall,  which  had  been  made  with  antiseptic  pre- 
cautions, was  closed.  Some  of  the  animals  so  treated  died  in  from  twelve 
hours  to  four  or  five  days,  while  others  survived  and  were  killed  on  the  sev- 
enth and  ninth  day. 

The  left  kidney,  especially  in  the  cases  which  survived  the  operation  for 
several  days,  was  found  to  be  two  or  three  times  as  large  as  the  right  and  to 
present  all  the  evidences  of  parenchymatous  inflammation.  The  pelvis  of 
the  kidney  contained  more  or  less  ammoniacal  urine,  pus,  and  bacilli ;  the 
parenchyma  gave  evidence  of  diffuse  inflammation  and  contained  numerous 
bacilli.  As  a  rule,  a  pure  culture  of  the  Bacillus  coli  communis  was  obtained 
from  the  inflamed  kidney. 

A  similar  experiment  was  made  with  a  species  of  proteus  (vulgaris  ?),  and 
with  a  similar  result.  The  animal  died  at  the  end  of  two  days.  The  left  kid- 
ney was  twice  as  large  as  the  right,  the  surface  of  a  deep-red  color  dotted 
with  numerous  white  spots ;  the  parenchyma  had  a  striped  appearance  on 
section  and  a  greenish  color  in  the  vicinity  of  the  pelvis,  which  contained 
ammoniacal  and  bloody  urine.  A  putrefactive  odor  was  given  off  from  the  or- 
gan. Proteus  in  pure  culture  was  recovered  from  the  interior  of  the  kidnev. 

Some  of  the  earlier  observers  have  described  non-liquefying  bacteria  ob- 
tained from  the  bladder  in  cases  of  chronic  cystitis  or  of  pyelonephritis  fol- 
lowing cystitis,  which,  according  to  Schmidt  and  Aschoff,  correspond  in 
morphological  and  biological  characters  with  Bacillus  coli  communis,  and 
were  no  doubt  identical  with  it.  They  believe  that  the  bacillus  described  by 
Clado  (1887)  under  the  name  of  "  Bacterie  septique,"  and  subsequently  found 
byAlbarran  and  Halle  in  forty-seven  out  of  fifty  cases  ^  of  cystitis  (fifteen 
times  in  pure  culture),  and  called  by  them  "Bacille  pyogene,"  is  in  fact  the 
Bacillus  coli  communis. 

Schmidt  and  Aschoff  say  that  the  changes  found  in  the  kidneys  of  rabbits 
after  the  injection  of  Bacillus  coli  communis  into  the  ligated  ureter  correspond 
with  those  seen  in  the  "surgical  kidney"  of  man.  They  were  surprised  at 
the  rapidity  with  which  the  bacilli  penetrated  the  urinary  tubules.  The  first 
changes  in  the  parenchyma  of  the  organ  occurred  at  the  end  of  thirty-six 
hours,  and  at  the  end  of  five  to  seven  days  these  changes  had  reached  their 
extreme  development.  They  evidently  depended  upon  the  invasion  of  the 
urinary  tubules  by  bacilli.  This  conclusion  corresponds  with  that  reached 
in  previous  researches  by  Albarran,  Achard  and  Renault,  and  by  Krogius. 

No  doubt  cystitis  and  ascending  pyelonephritis  are  usually  caused  by 
microorganisms  introduced  through  the  urethra  into  a  bladder  which  is  ren- 
dered susceptible  to  infection  by  mechanical  violence  or  chemical  irritation. 
The  most  frequent  cause  of  such  local  infection  is  the  Bacillus  coli  communis, 
which  is  constantly  present  in  the  intestine  and  upon  the  external  surface  in 
the  vicinity  of  the  anus,  from  whence  it  may  easily  be  transported  to  the  in- 
terior of  the  bladder  by  catheters,  etc. ,  used  by  the  patients  themselves  or  by 
their  medical  attendants. 


610  BACTERIA   IN  INFECTIOUS  DISEASES. 

PYOSALPINX. 

The  researches  of  Zweifel  (1892)  show  that  a  certain  proportion  of 
the  cases  of  pyosalpinx  are  due  to  the  presence  of  the  gonococcus;  in 
other  cases  the  infectious  agent  is  Streptococcus  pyogenes,  and  in  a 
few  cases  the  micrococcus  of  pneumonia  has  been  found  in  pus  from 
the  tubes  removed  by  operation.  In  seventy-one  cases  of  pyosalpinx 
or  of  salpingo-oophoritis,  which  were  examined  by  Zweifel  after  oper- 
ation, the  gonococcus  was  found  eight  times  and  streptococci  three 
times,  while  in  one  the  micrococcus  of  pneumonia  was  present. 
Menge  (1892)  found  gonococci,  and  no  other  microorganisms,  in 
three  cases.  Zweifel  believes  that  in  many  cases  in  which  the 
gonococcus  is  not  found  it  was  the  infectious  agent  to  which  the  in- 
flammation and  pus  formation  was  due,  but  that  its  presence  can 
only  be  demonstrated  in  recent  cases,  as  it  soon  dies  out. 

RELAPSING   FEVER. 

Due  to  infection  by  Spirillum  Obermeieri  (No.  191). 

RHEUMATIC    FEVER. 

The  symptoms  and  complications  of  acute  rheumatism  indicate 
that  it  is  an  infectious  disease,  and  the  researches  of  bacteriologists 
give  some  support  to  this  view.  Singer  (1895)  in  seventeen  cases 
investigated  found  Staphylococcus  pyogenes  albus  in  the  urine  in 
ten,  and  in  two  cases  in  the  blood;  in  three  cases  Streptococcus  pyo- 
genes was  found  in  the  urine  alone  and  in  two  cases  in  association 
with  Staphylococcus  pyogenes  albus;  in  one  case  Staphylococcus 
pyogenes  aureus  was  obtained  from  the  urine,  and  in  one,  compli- 
cated with  cystitis,  Bacillus  coli  communis.  According  to  Singer  the 
microorganisms  found  were  in  large  numbers  during  the  acute  stage 
of  the  disease  and  disappeared  when  convalescence  was  established. 

On  the  contrary,  Chvostek  (1895)  failed  to  find  microorganisms 
in  the  urine  in  nine  out  of  twelve  cases  examined  by  him;  in  three 
cases  he  obtained  micrococci — Staphylococcus  pyogenes  albus  in  one, 
Diplococcus  ureas  in  one,  and  an  undetermined  coccus  in  one.  The 
same  author  reports  that  in  numerous  examinations  of  the  contents 
of  the  inflamed  joints,  both  in  acute  and  chronic  cases,  he  failed  to 
find  bacteria  of  any  kind.  Sahli  (1892)  refers  to  the  uniformly  nega- 
tive results  obtained  by  different  bacteriologists  who  have  made  cul- 
tures from  fluid  obtained  from  the  inflamed  joints,  and  reports  a  fatal 
case  in  which  he  also  failed  to  obtain  cultures  from  the  fluid  in  the 
affected  joints,  but  in  which  Staphylococcus  pyogenes  citreus  was 
obtained  in  cultures  from  the  blood,  the  sy  no  vial  membrane  of  the 


BACTERIA  IN  INFECTIOUS  DISEASES.  611 

affected  joints,  the  fibrinous  exudate  upon  the  pericardium,  the  endo- 
cardial  vegetations,  and  the  swollen  bronchial  glands.  It  is  evident 
that  in  this  case  there  was  a  general  infection,  and  it  seems  probable 
that  the  joint  inflammation  was  due  to  the  same  microorganism  as 
that  of  the  other  tissues  involved  in  the  infectious  process.  It  is  pos- 
sible, however,  that  the  so-called  complications  of  acute  rheumatism 
result  from  a  mixed  infection.  Saint-Germain  (1893),  in  inoculation 
experiments  made  with  attenuated  cultures  of  staphylococci  into  the 
circulation  in  young  animals,  was  able  to  produce  joint  inflamma- 
tions with  effusion  of  serum  into  the  joint  cavity,  but  this  serum 
proved  to  be  sterile.  In  cases  of  subacute  or  chronic  articular  rheu- 
matism Bouchard  and  Charrin  report  (1891)  that  they  have  fre- 
quently obtained  cultures  of  Staphylococcus  pyogenes  albus  from  fluid 
drawn  from  the  affected  joints.  Sacaze  (1894)  calls  attention  to  the 
fact  that  acute  rheumatism  is  sometimes  preceded  by  a  lesion  through 
which  general  infection  may  have  occurred,  and  he  records  a  case  fol- 
lowing a  suppurating  wound,  from  the  pus  of  which  Staphylococcus 
pyogenes  albus  was  obtained  in  pure  culture,  also  several  cases  occur- 
ring in  individuals  with  hypertrophy  of  the  tonsils,  in  which  he 
supposes  that  this  was  the  channel  of  infection. 

If,  as  appears  probable,  acute  rheumatism  is  due  to  infection  by 
the  ordinary  pus  cocci,  we  may  suppose  that  this  occurs  in  conse- 
quence of  a  loss  of  the  natural  immunity  which  in  a  normal  condi- 
tion of  health  protects  man  from  invasion  by  these  micrococci,  which 
are  constantly  present  (especially  Staphylococcus  pyogenes  albus) 
upon  the  surface  of  the  body  and  of  mucous  membranes.  In  the 
writer's  work  on  "  Immunity,  Protective  Inoculations,  and  Serum- 
Therapy  "  (1895)  the  following  conclusion  is  reached  with  reference 
to  the  explanation  of  natural  immunity : 

"  The  experimental  evidence  submitted  considered  in  connection 
with  the  extensive  literature  relating  to  "phagocytosis,"  leads  us  to 
the  conclusion  that  natural  immunity  is  due  to  a  germicidal  sub- 
stance present  in  the  blood  serum  which  has  its  origin  (chiefly  at 
least)  in  the  leucocytes  and  is  soluble  only  in  an  alkaline  medium." 
Now,  in  acute  rheumatism  there  is  an  excess  of  acid  in  the  system, 
and  it  seems  quite  probable  that,  as  a  result  of  this,  the  natural  im- 
munity against  infection  by  these  micrococci  is  neutralized. 

RHINITIS  FIBRINOSA. 

In  a  considerable  number  of  cases  of  fibrinous  rhinitis,  reported  by  vari- 
ous authors,  the  Klebs-Lb'ffler  diphtheria  bacillus  has  been  demonstrated  to 
be  present — Baginsky,  Park,  Abbott,  Stamm,  Concetti,  Gerber,  and  Podack 
(1895),  and  others.  V ery  curiously  the  diphtheritic  process,  as  a  rule,  does 
not  extend  to  the  fauces,  and  the  disease  runs  a  favorable  course,  although 
virulent  diphtheria  bacilli  may  be  obtained  from  the  fibrinous  exudate. 


612  BACTERIA   IN   INFECTIOUS   DISEASES. 


RHINOSCLEROMA. 

This  appears  to  be  a  localized  infectious  process,  due  to  the  presence  of  the 
Bacillus  of  rhinoscleroma  (No.  58). 

SCARLET   FEVER. 

The  specific  infectious  agent  in  scarlet  fever  has  not  been  demonstrated. 
In  the  diphtheritic  exudate  frequently  seen  in  the  angina  of  scarlet  fever  a 
streptococcus  is  commonly  found  which  appears  to  be  identical  with  Strep- 
tococcus pyogenes;  and  in  the  secondary  affections  which  occur  in  the 
course  of  this  disease  or  during  convalescence,  when  suppuration  occurs,  one 
or  the  other  of  the  common  pyogenic  micrococci  is  usually  found  and  is 
doubtless  the  cause  of  the  local  inflammatory  process.  (See  Otitis  media.) 

Crajkowski  (1895)  has  reported  that  he  found  in  fifteen  cases,  in  which  he 
examined  the  blood  of  scarlet  fever  patients,  a  diplococcus  present  in  com- 
paratively small  numbers — seldom  more  than  one  or  two  in  a  microscopic 
field.  This  diplococcus  does  not  stain  by  Gram's  method  and  in  general  stains 
feebly  and  quickly  loses  its  color.  Cultures  were  obtained  in  bouillon  and 
upon  solid  media  (in  the  incubating  oven  ?),  but  not  in  gelatin.  The  develop- 
ment is  said  to  be  slow,  and  the  colonies  resemble  small  drops  of  dew — not 
more  than  one-third  to  one-half  of  a  millimetre  in  diameter.  Pathogenic  for 
mice,  but  not  for  rabbits.  Crajkowski  does  not  claim  that  the  etiologk-al 
relation  of  this  diplococcus  to  scarlet  fever  has  been  demonstrated.  His  dried 
blood  preparations  were  stained  by  the  method  of  Chencinsky. 

SCORBUTIS. 

In  an  epidemic  of  scurvy  in  a  cavalry  regiment  Babes  (1894)  found  in 
every  case,  in  the  necrotic  margin  of  the  mucous  membrane  of  the  gums,  a 
slender,  pointed,  and  bent  bacillus,  resembling  the  tubercle  bacillus  ;  this  did 
not  stain  by  Gram's  method.  This  bacillus  grew  in  nutrient  agar  at  37  C., 
and  the  cultures  injected  into  rabbits  caused  a  hemorrhagic  septicaemia  and 
death. 

SEPTICAEMIA. 

Septicaemia  in  man  is  usually  due  to  infection,  through  a  wound 
or  mucous  membrane  denuded  of  its  epithelium,  by  a  virulent  variety 
of  one  of  the  common  pus  cocci — Streptococcus  pyogenes  (No.  5),  or 
Staphylococcus  pyogenes  aureus  (No.  1).  Canon  (1894)  has  reported 
the  results  of  his  bacteriological  researches  in  seventy  cases  of  "septi- 
caemia, pyaemia,  and  osteomyelitis."  He  divides  his  cases  into  three 
groups.  In  the  first  group  ('20)  microorganisms  were  present  in  the 
blood  without  metastases,  in  the  second  (20)  microorganisms  were 
present  and  metastatic  foci  of  infection  were  found;  in  the  third 
group  there  were  metastatic  foci  but  no  microorganisms  were  found 
in  the  blood.  In  the  first  group  of  cases,  in  blood  obtained  post 
mortem  from  a  vein  in  the  arm,  streptococci  were  found  in  a  ma- 
jority of  the  cases,  staphylococci  in  a  smaller  number,  the  pneumonia 
coccus  in  one,  and  Bacillus  coli  communis  in  one.  In  this  group  the 
blood  was  examined  in  seven  cases  during  life  and  in  three  of  them 
with  a  positive  result.  In  eleven  cases  of  various  origin,  in  which 


BACTERIA   IN   INFECTIOUS   DISEASES.  613 

metastatic  foci  were  found,  streptococci  or  staphylococci  were  usu- 
ally found  in  the  blood  post  mortem,  and  in  four  out  of  five  cases  the 
blood  was  examined  during  life  with  a  positive  result.  In  five 
cases  of  osteomyelitis  blood  examinations  showed  that  Staphylococ- 
cus  pyogenes  aureus  was  usually  present.  (See  also  Osteoymelitis.) 

Petruschky  (1894)  in  his  extended  researches  obtained  positive 
results  in  seventeen  cases  in  which  the  blood  was  examined  during 
life  (eight  non-fatal  cases).  Streptococci  were  found  in  fourteen  of 
these  cases,  Staphylococcus  pyogenes  aureus  in  two,  and  Staphylococ- 
cus  pyogenes  albus  in  one.  In  puerperal  septicaemia  Streptococcus 
pyogenes  is  the  usual  infectious  agent.  (See  Puerperal  Fever.) 

In  gangrenous  septicaemia  (septicemie  gangreneuse  or  gazeuse  of 
French  authors)  the  bacillus  of  malignant  oedema  (No.  186)  is  the 
usual  infectious  agent,  but  this  is  a  localized  infectious  process  rather 
than  a  general  blood  infection. 

Certain  cases  of  so-called  purpura  haemorrhagica  are  probably 
due  to  general  blood  infection  by  pathogenic  bacilli  (see  Bacillus  of 
Tizzoni  and  Giovannini,  No.  145,  Bacillus  of  Babes,  No.  146,  and  Ba- 
cillus of  Kolb,  No.  147),  and  von  Dungern  has  described  a  case  of 
haemorrhagic  septica3mia  in  a  new-born  child  due  to  infection  by  a 
capsule  bacillus  (No.  164). 

Septiccemia  in  cattle  (Rinderseuche)  is  due  to  infection  by  Bacil- 
lus septica3mia3  baemorrhagicae  (No.  61),  as  is  also  septicaemia  in  deer 
(Wildseuche),  in  swine  (Schweineseuche),  and  in  rabbits  (Kaninchen- 
septikamie,  Koch) .  The  same  bacillus  is  the  cause  of  the  infectious 
disease  of  fowls  known  as  "  fowl  cholera. ' '  Other  bacteria  producing 
septicaemia  in  fowls  are  bacillus  of  Lucet  (No.  87)  and  Bacillus 
gallinarum  of  Klein  (No.  77).  Septiccemia  in  ducks  is  caused  by 
the  bacillus  of  Cornil  and  Toupet  (No.  62) ;  in  geese  by  Spirillum 
anserum  (No.  192) ;  in  frogs  by  Bacillus  hydrophilus  fuscus  (No. 
81);  in  fish  by  Bacillus  piscicidus  agilis  (No.  167),  and  Bacillus  of 
Emmerich  and  Weibel  (No.  169) ;  in  grouse  by  Bacillus  of  grouse 
disease  (No.  76),  in  parrots  by  Streptococcus  perniciosus  psittacorum 
(No.  43) ;  in  mice  by  numerous  bacteria,  including  Bacillus  erysipel- 
atos  suis  (No.  67),  Bacillus  typhi  murium  (No.  84),  Bacillus  of  Laser 
(No.  83),  and  Bacillus  of  Mereshkowski  (No.  168);  in  rabbits  by 
very  many  pathogenic  bacteria  from  various  sources  including  Bacil- 
lus septicaemia  haemorrhagicae  (No.  61),  Micrococcus  pneumoniae 
crouposae  (No.  8),  Bacillus  anthracis  (No.  45),  Bacillus  cuniculicida 
Havaniensis  (No.  93),  Bacillus  leporis  lethalis  (No.  94) ;  in  swine 
by  Bacillus  septicaemiae  haemorrhagicae  (No.  61),  Bacillus  of  swine 
plague,  Marseilles  (No.  65),  and  Bacillus  of  hog  cholera  (No.  63). 


614  BACTERIA  IN   INFECTIOUS   DISEASES. 

SILKWORMS,   INFECTIOUS    DISEASES  OF. 
See  Streptococcus  bombycis  (No.  24)  and  Nozema  bombycis  (No.  25). 

STOMATITIS. 

Schimmelbusch  (1889),  Lingard  (1888)  and  Foote  (1893)  have  described 
bacilli  obtained  by  them  from  the  necrotic  tissues  in  cases  of  iioma.  The 
bacillus  of  Lingard,  obtained  from  five  cases,  appears  to  be  identical  with 
that  of  Schimmelbusch  (No.  110).  In  the  case  reported  by  Foote  the  bacilli 
found  differed  from  the  bacillus  of  Schimmelbusch  as  shown  by  the  fact  that 
they  stained  by  Gram's  method. 

SYMPTOMATIC  ANTHRAX. 

Symptomatic  anthrax  ("blackleg,"  "quarter  evil,"  Ger.,  Rausch- 
brand)  is  due  to  infection  by  an  anaerobic  bacillus  (see  Bacillus  of 
Symptomatic  anthrax,  No.  188). 

SYPHILIS. 

The  etiology  of  syphilis  has  not  been  determined  by  the  researches  of 
bacteriologists.  For  an  account  of  the  microorganisms  which  have  been  en- 
countered in  syphilitic  lesions  the  reader  is  referred  to  the  article  on  the 
Bacillus  of  Lustgarten  (No.  55). 

TETANUS. 

Due  to  infection  by  Bacillus  tetani  (No.  185). 

OF  CATTLE. 

Billings  (1888)  has  announced  the  discovery  of  a  bacillus  in  the  blood  of 
cattle  suffering  from  Texas  fever,  which  he  supposed  to  be  the  cause  of  this 
disease,  but  the  investigations  of  other  bacteriologists  have  failed  to  confirm 
the  alleged  discovery.  It  appears  probable  that  a  mistake  in  diagnosis  was 
made,  and  that  the  disease  studied  by  Billings  was  an  infectious  form  of  sep- 
ticaemia in  cattle  similar  to  the  Kinderseuche  of  German  authors.  The  mi- 
croorganism which  he  has  described  as  coming  from  the  blood  of  the 
infected  animals  resembles  in  its  morphology  Bacillus  septicaemia'  li;i -UK >r- 
rhagicse  (No.  61),  and,  if  not  identical  with  this  widely  distributed  species, 
appears  to  be  very  nearly  related  to  it. 

The  researches  of  Smith  and  other  bacteriologists  connected  with  the 
United  States  Department  of  Agriculture  appear  to  have  elucidated  the  etiol- 
ogy of  this  disease,  and  to  show  that  it  is  due  to  a  blood  parasite  belonging 
to  the  protozoa  (Pyrosoma  bigeminum  of  Smith). 

TRACHOMA. 

Fuchs  (1894)  as  a  result  of  his  investigations  arrives  at  the  con- 
clusion that  trachoma  is  frequently  due  to  infection  by  the  gono- 
coccus.  He  believes  that  in  acute  cases  the  transfer  of  the  infectious 
secretions  causes  an  acute  gonorrhceal  ophthalmia;  and  that  when 


BACTERIA    IN   INFECTIOUS    DISEASES.  615 

this  becomes  chronic  a  similar  transfer  of  infectious  material  gives 
rise  to  the  chronic  inflammatory  process  known  as  trachoma.  Hoor 
(1895)  also  ascribes  trachoma  to  infection  by  gonococci,  and  believes 
that  in  their  etiology  papillary  trachoma  (blennorrhcea  chronica)  and 
granular  trachoma  (trachoma  verum)  are  identical. 

TYPHOID    FEVER. 

The  etiological  relation  of  Bacillus  typhi  abdominalis  (No.  46)  to 
typhoid  fever  is  now  generally  admitted  by  pathologists  and  bacteri- 
ologists. 

TYPHUS   FEVER. 

The  etiology  of  typhus  fever  has  not  been  determined  in  a  definite  man- 
ner. Hlava  (1888)  has  described  a  "  streptobacillus  "  which  he  supposes  to 
be  concerned  in  the  etiology  of  this  extremely  contagious  disease  ;  but  it  lias 
been  shown  by  other  investigators  that  this  bacillus  is  not  constantly  present, 
and  there  is  no  satisfactory  evidence  that  it  is  the  specific  infectious  agent. 

Thoinet  and  Calmette  (1892)  examined  the  blood  in  seven  cases  and  were 
unable  to  find  the  streptobacillus  of  Hlava  ;  their  cultures  from  the  spleen  of 
living  patients  and  from  the  spleen  and  blood  from  the  heart  of  recent  cadav- 
ers gave  a  negative  result.  They  found,  however,  in  blood  examined  under 
the  microscope  abnormal  elements,  in  the  form  of  motile  granules  and  fila- 
ments, which  were  sometimes  adherent  to  the  red  blood  corpuscles.  Lewa- 
schoff  in  1892  claimed  to  have  found  in  the  blood  of  typhus  patients  motile 
micrococci  having  long  spiral  flagella.  In  a  subsequent  epidemic  Lewa- 
schoff  (1894)  claims  to  have  found  the  same  microorganism  in  one  hundred 
and  eighteen  cases  examined,  and  also  to  have  obtained  in  cultures  from  blood 
taken  from  the  spleen  or  from  the  finger  the  same  microorganism,  sometimes 
solitary  and  provided  with  flagella,  and  sometimes  in  chains.  Weinshal 

(1892)  in  ten  cases  examined  by  the  method  recommended  by  Lewaschoff  was 
unable  to  find  the  microorganism  described  by  him  or  any  other.     Hlava 

(1893)  in  his  more  recent  researches  has  not  obtained  his  streptobacillus  in 
cultures  made  soon  after  death  (ten  cases) ;  but  he  obtained  various  micro- 
organisms in  his  cultures  from  the  spleen,  lungs,  etc.,  which  he  concludes 
are  not  directly  concerned  in  the  etiology  of  the  disease — streptococci,  staphy- 
lococci,  pseudo-diphtheria  bacilli,  and  a  proteus  ("Vibrio  proteus  ruber"). 
He  also  observed  in  the  blood  and  spleen  bodies  resembling  the  spores  of 
yeast  fungi. 

Dubieff  and  Bruhl  (1893)  in  nine  cases  examined  (six  post-mortem)  found 
in  small  numbers  in  the  blood  and  spleen  a  diplococcus,  called  by  them  Dip- 
lococcus  exaiithematicus.  This  is  said  to  have  been  difficult  to  cultivate  and 
to  have  been  present  in  enormous  numbers  in  mucus  from  the  nose  and 
throat  and  from  pneumonic  foci  in  the  lungs. 

Calmette  (1893),  in  a  detailed  account  of  the  microorganism  previously 
described  by  himself  and  Thoinet,  reports  that  this  is  especially  abundant  in 
splenic  pulp  obtained  by  an  aspirating  syringe  during  life.  The  bodies  found 
are  actively  motile,  and  from  2  to  3  /^  in  diameter,  having  sometimes  a  fili- 
form appendage  from  4  to  5  \i  in  length.  Long  spiral  filaments  are  also  occa- 
sionally seen  ;  these  are  from  20  to  30  \L  long  and  1  to  2  fj.  thick  ;  they  are  ac- 
tively motile.  Cultures  from  blood  were  obtained  in  media  containing  sugar 
or  acidified  with  lactic  or  tartaric  acid.  This  microorganism  is  believed  by 
Calmette  to  be  a  microscopic  fungus,  belonging  to  the  ascomycetes,  or  to  the 
genus  Ustilago.  Curtis  and  Combemale  (1893)  in  cultures  from  blood  drawn 
from  the  finger,  in  twelve  cases,  had  invariably  a  negative  result ;  in  three 


616  BACTERIA   IN   INFECTIOUS   DISEASES. 

cases  out  of  six  in  which  cultures  were  made  from  material  obtained  from 
the  spleen,  post  mortem,  a  very  minute  diplococcus  developed,  at  37  C.  This 
formed  a  grayish  layer  upon  the  surface  of  nutrient  agar  at  the  end  of  two 
or  three  days. 

TUBERCULOSIS. 

The  various  forms  of  tubercular  infection  in  man  are  due  to  a 
single  specific  infectious  agent — Bacillus  tuberculosis  (No.  53).  Tu- 
berculosis in  cattle  is  due  to  infection  by  the  same  bacillus.  The 
bacillus  which  produces  tuberculosis  in  fowls  closely  resembles  that 
of  human  tuberculosis,  but  owing  to  slight  differences  is  described 
under  a  separate  heading — Bacillus  tuberculosis  gallinarum  (No.  54). 

VARICELLA. 

Various  microorganisms  have  been  found  in  the  contents  of  the  vesicles 
and  pustules  of  varicella,  but  there  is  no  evidence  that  any  one  of  these  bears 
an  etiological  relation  to  this  specific  eruptive  fever. 

VARIOLA  AND   VACCINIA. 

The  etiology  of  small-pox  still  remains  undetermined.  The  common  pus 
cocci  and  various  other  microorganisms  are  found  in  the  characteristic  pus- 
tular eruption,  and  various  microorganisms  have  been  isolated  from  vaccine 
vesicles  ;  but  no  one  of  these  has  been  shown  to  possess  the  specific  pathogenic 
power  of  unfiltered  lymph  from  the  same  source.  The  experiments  of  Cars- 
tens  and  Coert  show  that  the  specific  virulence  of  vaccine  lymph  is  destroyed 
by  ten  minutes'  exposure  to  a  temperature  of  54°  C.  And  the  writer's  experi- 
ments show  that  various  disinfecting  agents  tested — chlorine,  sulphur  dioxide, 
nitrous  acid — destroy  the  infective  virulence  of  lymph  dried  upon  ivory 
points  in  about  the  same  proportion  as  is  required  for  the  destruction  of  some 
of  the  best-known  pathogenic  bacteria.  But  this  does  not  prove  that  viru- 
lence depends  upon  the  presence  of  a  living  microrganism,  however  probable 
this  appears,  for  certain  toxalbumins  are  likewise  destroyed  by  a  correspond- 
ingly low  temperature  and  by  the  action  of  various  chemical  disinfectants. 

Nikolsky  (1892)  obtained  from  the  base  of  the  pustules  of  small-pox  a  mo- 
tile, liquefying,  spore-producing  bacillus  which  when  introduced  into  ilie 
peritoneal  cavity  of  rabbits  is  said  to  have  given  rise  to  a  pustular  eruption  ; 
the  bacillus  was  recovered  in  cultures  from  these  pustules.  Grigorijew 
(1889)  in  three  cases  found  a  small  bacillus,  twice  as  long  as  thick,  which 
slowly  liquefied  gelatin,  and  did  not  coagulate  milk.  Besser  (1893)  givrs  an 
account  of  the  microorganisms  found  in  the  pustules  of  variola  and  adds  to 
the  list  a  bacillus  found  by  himself  in  a  single  case.  This  does  not  grow  in 
gelatin  at  the  room  temperature  and  is  more  slender  than  the  bacillus  of 
Grigoriew. 

The  results  of  the  researches  of  Martin  (1891)  have  been  reported  by  Ernst 
(1893).  He  obtained  various  bacteria  from  the  lymph  of  vat-cine  vesicles, 
and  among  these  was  a  bacillus  which  he  believed  to  be  the  specific  infectious 
agent.  This  he  was  able  to  cultivate  upon  the  surface  of  sterilized  blond 
serum  of  the  ox,  at  37°  C.,  and  his  cultures  of  the  ninth  generation  are  said 
to  have  produced  typical  cow-pox  when  inoculated  upon  calves.  He  says  : 
4 'The  material  'takes'  with  the  same  certainty  as  the  lymph  from  the  vesicle. 


BACTERIA   IN   INFECTIOUS   DISEASES.  617 

pure,  but  contained  quite  a  variety  of  bacteria.  In  every  instance  the  blood 
serum  was  liquefied."  As  to  the  morphology  he  says  :  "The  bacterium  va- 
ries in  form  according1  to  the  various  conditions  of  its  nutritive  environment 
and  the  consequent  rate  of  its  development.  The  most  constant  and  preva- 
lent form  is  a  short,  fine  bacillus  with  rounded  or  nearly  square  ends.  Those 
parts  of  the  culture  where  the  nutriment  is  apparently  exhausted  show  the 
same  "bacilli  in  short  chains,  longer  bacilli,  and  bacilli  much  enlarged  at  one 
end  or  the  middle,  as  if  in  preparation,  for  spore  formation."  As  Martin's  cul- 
tures were  not  pure  we  have  no  evidence  that  his  successful  inoculations 
were  due  to  the  particular  bacillus  which  attracted  his  attention.  Possibly  a 
microorganism  of  another  class  was  also  present  and  was  carried  over  from 
one  culture  to  another.  Ruete  and  Enoch  (1893)  have  also  reported  success- 
ful vaccinations  in  the  calf  with  a  micrococcus  which  they  cultivated  from 
vaccine  lymph. 

Buttersack  (1893)  as  a  result  of  his  researches  arrived  at  the  conclusion 
that  there  are  numerous  minute  elements  in  vaccine  lymph  which  do  not 
stain  and  are  sometimes  arranged  in  chains.  The  subsequent  researches  of 
Landmann  (1894)  and  others  indicate  that  the  supposed  microorganisms  of 
Buttersack  are  non-living,  albuminoid  granules,  artificially  produced  by  his 
method  of  investigation.  This  view  is  confirmed  by  the  investigations  of 
Draer  (1894). 

Guarnieri  (1892),  Monti  (1894),  Piana  and  Galli-Valerio  (1894),  and  Clarke 
(1895),  have  observed  amoeboid  microorganisms  in  the  pustules  of  variola  and 
in  vaccine  lymph  which  may  prove  to  be  the  specific  infectious  agent  in  this 
disease.  These  are  described  by  Guarnieri  under  the  name  Cytorycetes 
variolas  and  Cytorycetes  vacciniae.  According  to  Clarke  these  amoeboid  para- 
sites belong  to  the  Sprozoa.  E.  Pfeiffer  (1895)  has  studied  this  parasite  by 
inoculations  into  the  cornea  of  rabbits,  guinea-pigs,  and  calves. 

WHOOPING-COUGH. 

No  satisfactory  demonstration  has  yet  been  made  of  the  specific  infectious 
agent  in  whooping-cough. 

Hitter  (1892)  has  obtained  from  the  nasal  and  bronchial  secretions  in  cases 
(eighteen)  of  whooping-cough  a  small  diplococcus  which  he  believes  to  be 
the  cause  of  the  disease.  This  is  aerobic,  stains  by  Gram's  method,  and 
grows  upon  nutrient  agar  in  the  incubating  oven — not  in  bouillon,  in  gelatin, 
or  011  potato. 

Cohn  and  Neumann  (1893)  in  18  cases  in  which  they  made  a  careful  re- 
search by  approved  methods  were  only  able  to  demonstrate  the  presence  of 
Ritter's  diplococcus  in  one;  in  two  other  cases  somewhat  similar  diplococci  were 
found.  The  bacillus  described  by  Afanassiew  in  1887  (No.  119)  was  also  seen 
occasionally,  but  was  evidently  not  the  specific  infectious  agent.  The  micro- 
organism most  constantly  found  was  a  streptococcus,  apparently  identical 
with  Streptococcus  pyogenes.  This  was  present  in  20  cases  out  of  25  exam- 
ined, and  in  12  of  these  it  was  obtained  from  bronchial  mucus  in  such  num- 
bers as  almost  to  constitute  a  pure  culture. 

YELLOW   FEVER. 

The  results  of  investigations  made  by  the  writer  in  Cuba  during  the  sum- 
mers of  1888  and  1889  are  given  in  the  following  summary  statement  from 
the  Transactions  of  the  Tenth  International  Medical  Congress  (Berlin,  1890) : 

Bacterial  Researches  in  Yellow  Fever. 

The  report  relates  to  investigations  made  in  Havana,  Cuba,  during  the 
summers  of  1888  and  1889,  in  Decatur,  Alabama,  during  the  autumn  of  1888, 

43 


618  BACTERIA   IN   INFECTIOUS   DISEASES. 

and  in  the  pathological  and  biological  laboratories  of  the  Johns  Hopkins 
University  during  the  winters  of  1888  and  1889. 

Forty-two  autopsies  were  made  in  typical  cases  of  yellow  fever  and  seven- 
teen autopsies  in  other  diseases  for  comparative  researches. 

Aerobic  and  anaerobic  cultures  were  made  from  the  blood,  the  liver,  the 
kidney,  the  urine,  the  stomach,  and  the  intestine. 

The  experimental  data  recorded  in  this  report  show  that: 

The  specific  infectious  agent  in  yellow  fever  has  not  been  demonstrated. 

The  most  approved  bacteriological  methods  fail  to  demonstrate  the  con- 
stant presence  of  any  particular  microorganism  in  the  blood  and  tissues  of 
yellow-fever  cadavers. 

The  microorganisms  which  are  sometimes  obtained  in  cultures  from  the 
blood  and  tissues  are  present  in  comparatively  small  numbers ;  and  the  one 
most  frequently  found  (Bacterium  coli  commune)  is  present  in  the  intestine 
of  healthy  individuals,  and  consequently  its  occasional  presence  cannot  have 
any  etiological  import. 

A  few  scattered  bacilli  are  present  in  the  liver,  and  probably  in  other  or- 
gans, at  the  moment  of  death.  This  is  shown  by  preserving  portions  of 
liver,  obtained  at  a  recent  autopsy,  in  an  antiseptic  wrapping. 

At  the  end  of  twenty-four  to  forty-eight  hours  the  interior  of  a  piece  of 
liver  so  preserved  contains  a  large  number  of  bacilli  of  various  species,  the 
most  abundant  being  those  heretofore  mentioned  as  occasionally  found  in 
fresh  liver  tissue,  viz. ,  Bacterium  coli  commune  and  Bacillus  cadaveris. 

Blood,  urine,  and  crushed  liver  tissue  obtained  from  a  recent  autopsy  are 
not  pathogenic  in  moderate  amounts  for  rabbits  or  guinea-pigs. 

Liver  tissue  preserved  in  an  antiseptic  wrapping  at  a  temperature  of  28° 
to  30°  C.,  for  forty  eight  hours,  is  very  pathogenic  for  guinea-pigs  when  in- 
jected subcutaneous!  y. 

This  pathogenic  power  appears  to  be  due  to  the  microorganisms  present 
and  to  the  toxic  products  developed  as  a  result  of  their  growth.  It  is  not 
peculiar  to  yellow  fever,  inasmuch  as  material  preserved  in  the  same  way 
at  comparative  autopsies,  in  which  death  resulted  from  accident  or  other 
diseases,  has  given  a  similar  result. 

Having  failed  to  demonstrate  the  presence  of  a  specific  "germ"  in  the 
blood  and  tissues,  it  seems  probable  that  it  is  to  be  found  in  the  alimentary 
canal,  as  is  the  case  in  cholera.  But  the  extended  researches  made,  and  re- 
corded in  the  present  report,  show  that  the  contents  of  the  intestines  of  yel- 
low-fever cases  contain  a  great  variety  of  bacilli,  and  not  a  nearly  pure  cul- 
ture of  a  single  species,  as  is  the  case  in  recent  and  typical  cases  of  cholera. 

Comparatively  few  liquefying  bacilli  are  found  in  the  faeces  discharged 
during  life,  or  in  the  intestinal  contents  collected  soon  after  death  from  yel- 
low-fever cadavers.  On  the  other  hand,  non-liquefying  bacilli  are  very 
abundant. 

The  one  most  constantly  and  abundantly  present  is  the  Bacterium  coli 
commune  of  Escherich. 

This  is  associated  with  various  other  bacilli,  some  of  which  are  strict 
anaerobics  and  some  facultative  anaerobics. 

Among  the  facultative  anaerobics  is  one — my  Bacillus  X — which  has  been 
isolated  by  the  culture  method  in  a  considerable  number  of  cases  and  may 
have  been  present  in  all.  This  bacillus  has  not  been  encountered  in  the 
comparative  experiments  made.  It  is  very  pathogenic  for  rabbits  when  in- 
jected into  the  cavity  of  the  abdomen. 

It  is  possible  that  this  bacillus  is  concerned  in  the  etiology  of  yellow  fever, 
but  no  satisfactory  evidence  that  this  is  the  case  has  been  obtained  by  experi- 
ments on  the  lower  animals,  and  it  has  not  been  found  in  such  numbers  as 
to  warrant  the  inference  that  it  is  the  veritable  infectious  agent. 

All  other  microorganisms  obtained  in  pure  cultures  from  yellow-fever 
cadavers  appear  to  be  excluded,  either  by  having  been  identified  with  known 
species,  or  by  having  been  found  in  comparative  researches  made  outside  of 


BACTERIA   IN   INFECTIOUS   DISEASES.  619 

the  area  of  yellow  fever  prevalence,  or  by  the  fact  that  they  have  only  been 
found  in  small  numbers  and  in  a  limited  number  of  cases. l 

Finally  we  remark  that  many  facts  relating  to  the  origin  and  extension 
of  yellow-fever  epidemics  give  support  to  the  inference  that  the  specific  in- 
fectious agent  is  present  in  the  dejecta  of  those  suffering  from  the  disease, 
and  that  accumulations  of  faecal  matter,  and  of  other  organic  material  of  ani- 
mal origin,  furnish  a  suitable  nidus  for  the  development  of  the  "germ" 
when  climatic  conditions  are  favorable  for  its  growth. 

It  may  be  that  such  a  nidus  is  essential,  and  that  the  culture  media 
usually  employed  by  bacteriologists  do  not  afford  a  suitable  soil  for  this  par- 
ticular microbe. 

It  is  also  possible  that  its  development  depends  upon  the  presence  of  other 
microorganisms  found  in  faecal  matter,  which  give  rise  to  chemical  products 
required  for  the  development  of  this  one. 

Some  of  the  microorganisms  present  in  the  dejecta  of  yellow-fever  pa- 
tients, as  shown  by  stained  smear  preparations,  have  not  developed  in  the 
cultures  made,  either  aerobic  or  anaerobic.  One  extremely  slender  filiform 
bacillus,  which  can  only  be  seen  with  high  powers  and  which  is  quite  abun- 
dant in  some  of  my  preparations,  has  never  been  obtained  in  the  cultures 
made,  and  no  doubt  there  are  others  in  the  same  category. 

That  the  yellow-fever  germ  is  a  strict  anaerobic,  or  that  it  will  only  grow 
in  a  special  nidus,  may  be  inferred  from  certain  facts  relating  to  the  exten- 
sion of  epidemics. 

There  is  rio  evidence  that  yellow  fever  is  propagated  by  contamination  of 
the  supply  of  drinking  water,  as  frequently,  and  probably  usually,  occurs  in 
the  case  of  typhoid  fever  and  cholera.  Moreover,  epidemics  extend  in  a 
more  deliberate  manner  and  are  restricted  within  a  more  definite  area  than 
is  the  case  with  cholera  and  typhoid  fever.  It  is  usually  at  least  ten  days  or 
two  weeks  after  the  arrival  of  an  infected  vessel  or  of  a  person  sick  with  the 
disease  before  cases  of  local  origin  occur ;  and  these  cases  occur  in  the  imme- 
diate vicinity  of  the  imported  case  or  infected  vessel.  When  the  disease  has 
effected  a  lodgment  the  area  of  infection  extends  slowly  and  usually  has 
well-defined  boundaries.  In  towns  and  cities  having  a  common  water  sup- 
ply one  portion  remains  perfectly  healthy,  while  another,  and  usually  the 
most  filthy  portion,  may  be  decimated  by  the  scourge. 

The  experimental  evidence  recorded,  and  the  facts  just  stated,  seem  to 
justify  the  recommendation  that  the  dejecta  of  yellow-fever  patients  should 
be  regarded  as  infectious  material,  and  that  such  material  should  never  be 
thrown  into  privy  vaults  or  upon  the  soil  until  it  has  been  completely  disin- 
fected. 

This  rule  thoroughly  enforced,  together  with  an  efficient  quarantine  ser- 
vice and  proper  attention  to  the  sanitary  police  of  our  exposed  seaport  cities, 
would,  I  believe,  effectually  prevent  this  pestilential  disease  from  again  ob- 
taining a  foothold  within  the  limits  of  the  United  States. 

1  The  possibility,  of  course,  remains  that  the  specific  infectious  agent  in  yellow 
fever  may  belong  to  an  entirely  different  class  of  microorganisms  from  the  bacteria, 
or  that  it  may  be  ultra-microscopic  or  not  capable  of  demonstration  in  the  tissues 
by  the  staining  methods  usually  employed  by  bacteriologists. 


PART   FOURTH. 


SAPEOPHYTES. 

I.  BACTERIA  IN  THE  AIR.    II.  BACTERIA  IN  WATER.    III.    BACTERIA  IN 
THE  SOIL.    IV.  BACTERIA  ON  THE  SURFACE  OF  THE  BODY  AND  OF  EX- 
POSED Mucous  MEMBRANES.    V.  BACTERIA  OF  THE  STOMACH  AND 
INTESTINE.    VI.  BACTERIA  OF  CADAVERS  AND  OF  PUTREFYING 
MATERIAL  FROM  VARIOUS  SOURCES.     VII.  BACTERIA 
IN  ARTICLES  OF  FOOD. 


BACTERIA  IN  THE  AIR. 

THE  saprophytic  bacteria  are  found  wherever  the  organic  material 
which  serves  as  their  pabulum  is  exposed  to  the  air  under  conditions 
favorable  to  their  growth.  The  essential  conditions  are  presence  of 
moisture  and  a  suitable  temperature.  The  organic  material  may  be 
in  solution  in  water  or  in  the  form  of  moist  masses  of  animal  or 
vegetable  origin,  and  the  temperature  may  vary  within  considerable 
limits — 0°  to  70°  C.  But  the  species  which  takes  the  precedence  will 
depend  largely  upon  special  conditions.  Thus  certain  species  multi- 
ply abundantly  in  water  which  contains  comparatively  little  organic 
pabulum,  and  others  require  a  culture  medium  rich  in  albuminous 
material  or  in  carbohydrates  ;  some  grow  at  a  comparatively  low  or 
high  temperature,  while  others  thrive  only  at  a  temperature  of  20°  to 
40°  C.  or  have  a  still  more  limited  range ;  some  require  an  abun- 
dant supply  of  oxygen,  and  others  will  not  grow  in  the  presence  of 
this  gas.  Our  statement  that  saprophytic  bacteria  are  found  wherever 
the  organic  material  which  serves  as  their  pabulum  is  exposed  to  the 
air — under  suitable  conditions — relates  to  the  fact  that  it  is  through 
the  air  that  these  bacteria  are  distributed  and  brought  in  contact 
with  exposed  material.  It  is  a  matter  of  common  laboratory  experi- 
ence that  sterilized  organic  liquids  quickly  undergo  putrefactive  de- 
composition when  freely  exposed  to  the  air,  and  may  be  preserved  in- 
definitely when  protected  from  the  germs  suspended  in  the  air  by 
means  of  a  cotton  air  filter.  But  the  organic  pabulum  required  for 
the  nourishment  of  these  bacteria  is  not  found  in  the  air  in  any  con- 
siderable amount,  and  if  they  ever  multiply  in  the  atmosphere  it 
must  be  under  very  exceptional  conditions.  Their  presence  is  due  to 
the  fact  that  they  are  wafted  from  surfaces  where  they  exist  in  a 
desiccated  condition,  and,  owing  to  their  levity,  are  carried  by  the 
wind  to  distant  localities.  But,  under  the  law  of  gravitation,  when 
not  exposed  to  the  action  of  currents  of  air  they  constantly  fall 
again  upon  exposed  surfaces,  which,  if  moist,  retain  them,  or  from 
which,  if  dry,  they  are  again  wafted  by  the  next  current  of  air. 
Under  these  circumstances  it  is  easy  to  understand  why,  as  deter- 


624 


BACTERIA   IN   THE   AIR. 


mined  by  investigation^  more  bacteria  are  found  near  the  surface  of 
the  earth  than  at  some  distance  above  the  surface,  more  over  the 
land  than  over  the  ocean,  more  in  cities  with  their  dust-covered 
streets  than  in  the  country  with  its  grass-covered  fields. 

Careful  experiments  have  shown  that  bacteria  do  not  find  their 
way  into  the  atmosphere  from  the  surface  of  liquids,  unless  portions 
of  the  liquid  containing  them  are  projected  into  the  air  by  some 
mechanical  means,  such  as  the  bursting  of  bubbles  of  gas.  Cultures 
of  pathogenic  bacteria  freely  exposed  to  the  air  in  laboratories  do  not 
endanger  the  health  of  those  who  work  over  them;  but  if  such  a  cul- 
ture is  spilled  upon  the  floor  and  allowed  to  remain  without  disin- 
fection, when  it  is  desiccated  the  bacteria 
contained  in  it  will  form  part  of  the  dust  of 
the  room  and  might  be  dangerous  to  its 
occupants.  Bacteria  do  not  escape  into  the 
air  from  the  surface  of  the  fluid  contents  of 
sewers  and  cesspools,  but  changes  of  level 
may  cause  a  deposit  upon  surfaces,  which 
is  rich  in  bacteria,  and  when  dried  this  ma- 
terial is  easily  carried  into  the  atmosphere 
by  currents  of  air. 

Tyndall's  experiments  (1869)  show  that 
in  a  closed  receptacle  in  which  the  air  is 
perfectly  still  all  suspended  particles  are  af- 
ter a  time  deposited  on  the  floor  of  the  closed 
air  chamber.  And  common  experience  de- 
monstrates the  fact  that  the  dust  of  the  at- 
mosphere is  carried  by  the  wind  from  ex- 
posed surf  aces  and  again  deposited  when  the 
air  is  at  rest.  This  dust  as  deposited,  for 
example,  in  our  dwellings  contains  innu- 
merable bacteria  in  a  desiccated  condition, 
and  the  smallest  quantity  of  it  introduced 
into  a  sterile  organic  liquid  will  cause  it  to 
undergo  putrefactive  decomposition,  and 
by  bacteriological  methods  it  will  be  found 
to  contain  various  species  of  bacteria.  Such 
dust  also  contains  the  spores  of  various 
mould  fungi  which  are  present  in  the  atmo- 
sphere, usually  in  greater  numbers  than  the 
bacteria.  The  mould  fungi  are  air  pL-snts 
which  vegetate  upon  the  surface  of  moist  organic  material  and  form 
innumerable  spores,  which  are  easily  wafted  into  the  air,  both  on 
account  of  their  low  specific  gravity  and  minute  size,  and  because  they 


FIG.  188.  —  Penicillum  glau- 
cum;  m,  mycelium,  from  which 
is  given  off  a  branching  pedicle 
bearing  spores.  X  160. 


BACTERIA   IN    THE   AIR.  625 

are  borne  upon  projecting  pedicles  by  which  they  are  removed  from 
the  moist  material  upon  which  and  in  which  the  mycelium  develops 
(Fig.  188),  and,  being  dry,  are  easily  carried  away  by  currents  of  air. 

Bacteriologists  have  given  much  attention  to  the  study  of  the  mi- 
croorganisms suspended  in  the  atmosphere,  with  especial  reference  to 
hygienic  questions.  The  methods  and  results  of  these  investigations 
will  be  considered  in  the  present  section. 

Pasteur  (1860)  demonstrated  the  presence  of  living  bacteria  in  the 
atmosphere  by  aspirating  a  considerable  quantity  of  air  through  a 
filter  of  gun-cotton  or  of  asbestos  contained  in  a  glass  tube.  By  dis- 
solving the  gun-cotton  in  alcohol  and  ether  he  was  able  to  demon- 
strate the  presence  of  various  microorganisms  by  a  microscopical  ex- 
amination of  the  sediment,  and  by  placing  the  asbestos  filters  in 
sterilized  culture  media  he  proved  that  living  germs  had  been  filtered 
out  of  the  air  passed  through  them. 


FIG.  189. 

A  method  employed  by  several  of  the  earlier  investigators  con- 
sisted in  the  collection  of  atmospheric  moisture  precipitated  as  dew 
upon  a  surface  cooled  by  a  freezing  mixture.  This  was  found  to  con- 
tain living  bacteria  of  various  forms.  The  examination  of  rain  water, 
which  in  falling  washes  the  suspended  particles  from  the  atmosphere, 
gave  similar  results. 

The  first  systematic  attempts  to  study  the  microorganisms  of  the 
air  were  made  by  Maddox  (1870)  and  by  Cunningham  (1873),  who 
used  an  aeroscope  which  was  a  modification  of  one  previously  de- 
scribed by  Pouchet.  In  the  earlier  researches  of  Miquel  a  similar 
aeroscope  was  used.  This  is  shown  in  Fig.  189.  The  opening  to  the 
cylindrical  tube  A  is  kept  facing  the  wind  by  means  of  a  wind  vane, 
and  when  the  wind  is  blowing  a  current  passes  through  a  small  aper- 
ture in  a  funnel-shaped  partition  which  is  properly  placed  in  the 
cylindrical  tube.  A  glass  slide,  upon  the  lower  surface  of  which  a 


626  BACTERIA   IN   THE   AIR. 

mixture  of  glycerin  and  glucose  has  been  placed,  is  adjusted  near  the 
opening  of  the  funnel,  at  a  distance  of  about  three  millimetres,  so 
that  the  air  escaping  through  the  small  orifice  is  projected  against  it. 
By  this  arrangement  a  considerable  number  of  the  microorganisms 
present  in  the  air,  as  well  as  suspended  particles  of  all  kinds,  are  ar- 
rested upon  the  surface  of  the  slide  and  can  be  examined  under  the 
microscope  or  studied  by  bacteriological  methods.  But  an  aeroscope 
of  this  kind  gives  no  precise  information  as  to  the  number  of  living 
germs  contained  in  a  definite  quantity  of  air.  The  microscopical  ex- 
amination also  fails  to  differentiate  the  bacteria  from  particles  of 
various  kinds  which  resemble  them  in  shape,  and  the  microorgan- 
isms seen  are  for  the  most  part  spores  of  various  fungi  mingled  with 
pollen  grains,  vegetable  fibres,  plant  hairs,  starch  granules,  and 
amorphous  granular  material. 

Another  method,  which  has  been  employed  by  Cohn,  Pasteur, 
Miquel,  and  others,  consists  in  the  aspiration  of  a  definite  quantity  of 
air  through  a  culture  liquid,  which  is  then  placed  in  an  incubating 
oven  for  the  development  of  microorganisms  washed  out  of  the  air 
which  has  been  passed  through  it.  This  method  shows  that  bacteria 
of  different  species  are  present,  but  gives  no  information  as  to  their 
relative  number,  and  requires  further  researches  by  the  plate  method 
to  determine  the  characters  of  the  several  species  in  pure  cultures. 

A  far  simpler  method  consists  in  the  exposure  of  a  solid  culture 
medium,  which  has  been  carefully  sterilized  and  allowed  to  cool  on  a 
glass  plate  or  in  a  Petri's  dish,  for  a  short  time  in  the  air  to  be  ex- 
amined. Bacteria  and  mould  fungi  deposited  from  the  air  adhere  to 
the  surface  of  the  moist  culture  medium,  and  form  colonies  when  the 
plate,  enclosed  in  a  covered  glass  dish,  is  placed  in  the  incubating  oven. 
The  number  of  these  colonies  which  develop  after  exposure  in  the 
air  for  a  given  time  enables  us  to  estimate  in  a  rough  way  the  num- 
ber of  microorganisms  present  in  the  air  of  the  locality  where  tin- 
exposure  was  made  ;  and  the  variety  of  species  is  determined  by  ex- 
amining the  separate  colonies,  each  of  which  is,  as  a  rule,  developed 
from  a  single  germ.  By  exposing  a  number  of  plates  at  different 
times  this  method  enables  us  to  determine  what  species  are  m<  >st 
abundant  in  a  given  locality  and  the  comparative  number  in  dif- 
ferent localities,  as  determined  by  counting  the  colonies  after  ex- 
posure for  a  definite  time — e.g.,  ten  minutes.  Of  course  we  will  only 
obtain  evidence  of  the  presence  of  such  aerobic  bacteria  as  will 
grow  in  our  culture  medium.  The  anaerobic  bacteria  may  be  studied 
by  placing  plates  exposed  in  a  similar  way  in  an  atmosphere  of  hydro- 
gen. Bacteria  which  grow  slowly  and  only  under  special  conditions, 
like  the  tubercle  bacillus,  would  be  likely  to  escape  observation,  as 
the  mould  fungi  and  common  saprophytes  would  take  complete  pos- 


BACTERIA  IN   THE   AIR. 


627 


session  of  the  surface  of  the  culture  medium  before  the  others  had 
formed  visible  colonies.  Students  will  do  well  to  employ  this  simple 
and  satisfactory  method  for  the  purpose  of  making  themselves  familiar 
with  the  more  common  atmospheric  organisms,  and  they  will  find 
the  shallow  glass  dishes  with  a  cover,  known  as  Petri's  dishes,  very 
convenient  for  the  purpose.  These  dishes  should  be  sterilized  in  the 
hot-air  oven  and  sufficient  sterile  nutrient  gelatin  or  agar  poured 
into  them  to  cover  the  bottom.  After  the  culture  medium  has  be- 
come solid  by  cooling,  the  exposure  may  be  made  by  simply  remov- 
ing the  cover  and  replacing  it  at  the  end  of  the  time  fixed  upon. 


FIG.  190. 

To  determine  in  a  more  exact  way  the  number  of  microorganisms 
contained  in  a  given  quantity  of  air  will  require  other  methods.  But 
we  may  say,  en  passant,  that  such  a  determination  is  usually  not  of 
great  scientific  importance.  The  number  is  subject  to  constant  fluc- 
tuations in  the  same  locality,  depending  upon  the  force  and  direction 
of  the  wind.  If  we  have  on  one  side  of  our  laboratory  a  dusty 
street  and  on  the  other  a  green  field,  more  bacteria  will  naturally  be 
found  when  the  wind  blows  from  the  direction  of  the  street  than 
when  it  comes  from  the  opposite  direction  ;  or,  if  the  air  is  filled  with 
dust  from  recently  sweeping  the  room,  we  may  expect  to  find  very 


628  BACTERIA   IN   THE   AIR. 

many  more  than  when  the  room  has  been  undisturbed  for  some  time. 
The  painstaking  researches  which  have  already  been  made  have  es- 
tablished in  a  general  way  the  most  important  facts  relating  to  the 
distribution  of  atmospheric  bacteria,  but  have  failed  to  show  any  de- 
finite relation  between  the  number  of  atmospheric  bacteria  and  the 
prevalence  of  epidemic  diseases.  In  the  apparatus  of  Hesse,  Fig. 
190,  a  glass  tube,  having  a  diameter  of  four  to  five  centimetres  and  a 
length  of  half  a  metre  to  a  metre,  is  employed.  In  use  this  is  sup- 
ported upon  a  tripod,  as  shown  in  the  figure,  and  air  is  drawn 
through  it  by  a  water  aspirator  consisting  of  two  flasks,  also  shown. 
The  upper  flask  being  filled  with  water,  this  flows  into  the  lower 
flask  by  siphon  action,  and  upon  reversing  the  position  of  the  flasks 
number  one  is  again  filled.  By  repeating  this  operation  as  many 
times  as  desired  a  quantity  of  air  corresponding  with  the  amount  of 
water  passed  from  the  upper  to  the  lower  flask  is  slowly  aspirated 
through  the  horizontal  glass  tube.  The  microorganisms  present  are 
deposited  upon  nutrient  gelatin  previously  allowed  to  cool  upon  the 
lower  portion  of  the  large  glass  tube.  The  air  enters  through  a  small 
opening  in  a  piece  of  sheet  rubber  which  is  tied  over  the  extremity 
of  the  horizontal  tube,  and  before  the  aspiration  is  commenced  this 
opening  is  covered  by  another  piece  of  sheet  rubber  tied  over  the 
first.  Experience  shows  that  when  the  air  is  slowly  aspirated  most 
of  the  germs  contained  in  it  are  deposited  near  the  end  of  the  tube 
through  which  it  enters.  The  colonies  which  develop  upon  the  nu- 
trient gelatin  show  the  number  and  character  of  living  microorgan- 
isms contained  in  the  measured  quantity  of  air  aspirated  through  the 
apparatus.  The  method  with  a  soluble  filter  of  pulverized  sugar,  to 
be  described  hereafter,  is  preferable  when  exact  results  are  desired; 
and  for  the  purpose  of  determining  the  relative  abundance  and  the 
variety  of  microorganisms  present  in  the  atmosphere  of  a  given  lo- 
cality the  exposure  of  nutrient  gelatin  in  Petri's  dishes  is  far  simpler, 
and,  as  a  rule,  will  furnish  all  the  information  that  is  of  real 
value. 

In  his  extended  researches  made  at  the  laboratory  of  Montsouri, 
in  Paris,  Miquel  has  used  various  forms  of  apparatus  and  has  i  >1  >- 
tained  interesting  results  ;  but  his  method  of  ensemencements  frafr 
tionnes  requires  a  great  expenditure  of  time  and  patience,  and  the 
more  recent  method  with  soluble  filters  is  to  be  preferred. 

In  his  latest  modification  of  the  method  referred  to  Miquel  used  a 
flask  like  that  shown  in  Fig.  191.  From  twenty  to  forty  cubic  cen- 
timetres of  distilled  water  are  introduced  into  this  flask.  The  cap  A 
contains  a  cotton  air  filter  and  is  fitted  to  the  neck  of  the  flask  by  a 
ground  joint.  This  is  removed  during  the  experiment.  The  tube  C 
is  connected  with  an  aspirator.  It  contains  two  cotton  or  asbestos 


BACTERIA   IN   THE   AIR. 


629 


Fio.  191. 


filters,  c  and  b.  The  cap  being  removed  and  the  aspirator  attached, 
the  air  is  drawn  through  the  water,  by  which  suspended  germs  are 
arrested  ;  or  if  not  they  are  caught  by  the  inner  cotton  plug  b.  The 
sealed  point  of  the  tube  B  is  now  broken  off,  and  the  contents  of  the 
flask  equally  divided  in  thirty  to  forty  tubes  containing  bouillon, 
which  are  placed  in  the  incubating  oven. 
Twenty-five  cubic  centimetres  of  bouillon 
are  also  introduced  into  the  flask,  and  the 
cotton  plug  b  is  pushed  into  it  so  that  any 
bacteria  arrested  by  it  may  develop.  If 
one-fourth  or  one-fifth  of  the  bouillon  tubes 
show  a  development  of  bacteria  it  is  in- 
ferred that  each  culture  originated  from 
a  single  germ,  and  the  number  present  in 
the  amount  of  air  drawn  through  the  flask 
is  estimated  from  the  number  of  tubes  in 
which  development  occurs. 

The  method  adopted  by  Straus  and  Wiirtz  is  more  convenient  and 
more  reliable  in  its  results.  This  consists  in  passing  the  air  by  means 
of  an  aspirator  through  liquefied  nutrient  gelatin  or  agar.  The  ap- 
paratus shown  in  Fig.  192  is  used  for  this  purpose.  Two  cotton 
plugs  are  placed  in  the  tube  B,  to  which  the  aspirator  is  attached, 
and  after  the  determined  quantity  of  air  has  been  passed  through  the 
liquefied  medium  the  inner  plug  is  pushed  down  with  a  sterilized 
platinum  needle  so  as  to  wash  out  in  the  culture 
medium  any  germs  arrested  by  it.  Finally  the 
gelatin  or  agar  is  solidified  upon  the  walls  of 
the  tube  A  by  rotating  it  upon  a  block  of  ice  or 
under  a  stream  of  cold  water.  It  is  now  put 
aside  for  the  development  of  colonies,  which  are 
counted  to  determine  the  number  of  germs  pre- 
sent in  the  quantity  of  air  passed  through  the 
liquefied  culture  medium.  The  main  difficulty 
with  this  apparatus  is  found  in  the  fact  that  the 
nutrient  gelatin  foams  when  air  is  bubbled 
through  it ;  for  this  reason  an  agar  medium  is 
to  be  preferred.  In  using  this  it  will  be  neces- 
sary to  place  the  liquefied  agar  in  a  bath  main- 
tained at  40°  C.  Foaming  of  the  gelatin  is  pre- 
vented by  adding  a  drop  of  olive  oil  before  ster- 
ilization in  the  steam  sterilizer.  But  this  inter- 
feres with  the  transparency  of  the  medium. 
In  the  earlier  experiments  upon  atmospheric  organisms  Pasteur 
used  a  filter  of  asbestos,  which  was  subsequently  washed  out  in  a 


FlO.  192. 


630 


BACTERIA   IN   THE   AIR. 


culture  liquid.     A  filter  of  this  kind  washed  out  in  liquefied  gelatin 
or  nutrient  agar  would  give  more  satisfactory  results,  as  the  culture 
medium  could  be  poured  upon  plates  or  spread  upon  the  walls  of  a 
test  tube  and  the  colonies  counted  in  the  usual  way.     Petri  prefers 
to  use  a  filter  of  sand,  which  he  finds  by  experiment  arrests  the  mi- 
croorganisms suspended  in  the  atmosphere,  and  which  is  subsequently 
distributed  through  the  culture  medium.     The  sand  used  is  such  as 
has  been  passed  through  a  wire  sieve  having 
openings  of  0. 5  millimetre  in  diameter.     This  is 
sterilized  by  heat,  and  is  supported  in  a  cylin- 
drical glass  tube  by  small  wire-net  baskets.    The 
complete  arrangement  is  shown  in  Fig.    193. 
Two  sand  filters,  cl  and  C2,  are  used,  the  lower 
one  of  which  serves  as  a  control  to  prove  that 
all  microorganisms  present  in  the  air  have  been 
arrested  by  the  upper  one.     The  upper  filter  is 
protected,  until  the  aspirator  attached  to  the 
tube  h  is  put  in  operation,  by  a  sterile  cotton 
plug,  not  shown  in  the  figure  which  represents 
the  filter  in  use.    Petri  uses  a  hand  air  pump  as 
an  aspirator,  and  passes  one  hundred  litres  of 
air  through  the  sand  in  from  ten  to  twenty 
minutes.     The  sand  from  the  two  filters  is  then 
distributed  in  shallow  glass  dishes  and  liquefied 
gelatin  is  poured  over  it ;  this  is  allowed  to  sol- 
idify and  is  put  aside  for  the  development  of 
colonies.    The  principal  objection  to  this  method 
is  the  presence  of  the  opaque  particles  of  sand 
in  the  culture  medium.    This  objection  has  been 
overcome  by  the  use  of  soluble  filters,  a  method 
first  employed  by  Pasteur  and  since  perfected 
by  Sedgwick  and  by  Miquel.     The  most  useful 
material  for  the  purpose  appears  to  be  cane 
sugar,  which  can  be  sterilized  in  the  hot-air  oven 
at  150°  C.  without  undergoing  any  change  in 
its  physical  characters.     Loaf  sugar  is  pulver- 
ized in  a  mortar  and  passed  through  two  sieves 
in  order  to  remove  the  coarser  grains  and  the 

very  fine  powder,  leaving  for  use  a  powder  having  grains  of  about 
one-half  millimetre  in  diameter.  This  powdered  sugar  is  placed 
in  a  glass  tube  provided  with  a  cap  having  a  ground  joint  and  a  cot- 
ton plug  to  serve  as  an  air  filter  (A,  Fig.  104),  or  in  a  tube  such  as  is 
shown  at  B,  having  the  end  drawn  out  and  hermetically  sealed.  Two 
cotton  plugs  are  placed  at  the  lower  portion  of  the  tube,  at  a  and  at  b. 


Fio.   193. 


BACTERIA   IN   THE   AIR. 


631 


Glass  tubing  having  a  diameter  of  about  five  millimetres  is  used  in 
making  these  tubes,  and  from  one  to  two  grammes  of  powdered  sugar 
is  a  suitable  quantity  to  use  as  a  filter.     The  whole  apparatus  is  steril- 
ized for  an  hour  at  150°  C.  in  a  hot-air  oven  after  the  pulverized 
sugar  has  been  introduced.     Before  using  'it  will  be  necessary  to 
pack  the  sugar  against  the  supporting  plug  a  by  gently  striking  the 
lower  end  of  the  tube,  held  in  a  vertical  position,  upon  some  horizon- 
tal surface ;    and  during  aspiration 
the  tube  must  remain  in  a  vertical          J\ 
position,  or  nearly  so,  in  order  that 
the  sugar  may  properly  fill  its  entire 
calibre.     The  aspirator  is  attached  to 
the  lower  end  of  the  tube  by  a  piece 
of  rubber  tubing.     When  the  tube  B 
is  used  the  sealed  extremity  is  broken 
off  at  the  moment  that  the  aspirator 
is  set  in  action,  and  it  is  again  sealed 
in  a  flame  after  the  desired  amount 
of  air  has  been  passed  through  the 
filter.     The  next  step  consists  in  dis- 
solving the  sugar  in  distilled  water 
or  in  liquefied  gelatin.      To  insure 
the  removal  of  all  the  sugar  the  cot- 
ton plug  a  may  be  pushed  out  with  a 
sterilized  glass  rod,  after  removing  b 
with  forceps.     From  fifty  to  five  hun- 
dred cubic  centimetres    of    distilled 
water,  contained  in  an  Erlenmeyer 
flask  and  carefully  sterilized,  may  be 
used,  the  amount  required  depending 
upon  circumstances  relating  to  the 
conditions  of    the  experiment.      By 
adding  five  or  ten  cubic  centimetres          01  &         KJ  1> 
of  this  water,  containing  the  sugar  FlG  194  FlG>  195 

and  microorganisms  arrested  by  it, 

to  nutrient  gelatin  or  agar  liquefied  by  heat,  and  then  making  Es- 
march  roll  tubes,  the  number  of  germs  in  the  entire  quantity  is  easily 
estimated  by  counting  the  colonies  which  develop  in  the  roll  tubes. 

Sedgwick  and  Tucker,  in  a  communication  made  to  the  Boston 
Society  of  Arts,  January  12th,  1888,  were  the  first  to  propose  the  use 
of  a  soluble  filter  of  granulated  sugar  for  collecting  atmospheric 
germs.  Their  complete  apparatus  consists  ,of  an  exhausted  receiver, 
from  which  a  given  quantity  of  air  is  withdrawn  by  means  of  an  air 
pump.  A  vacuum  gauge  is  attached  to  the  receiver,  which  is  coupled 


632  BACTERIA   IN   THE  AIR. 

with  the  glass  tube  containing  the  granulated -sugar  filter  by  a  piece 
of  rubber  tubing.  Instead  of  transferring  the  soluble  filter  to  gela- 
tin in  test  tubes,  they  use  a  large  glass  cylinder  having  a  slender 
stem,  in  which  the  sugar  is  placed  (Fig.  195).  After  the  aspiration 
liquefied  gelatin  is  introduced  into  the  large  glass  cylinder,  which  is 
held  in  a  horizontal  position  ;  the  sterilized  cotton  plug  is  then  re- 
placed in  the  mouth  of  the  cylinder,  the  sugar  is  pushed  into  the 
liquefied  gelatin  and  dissolved,  and  by  rotating  the  cylinder  upon  a 
block  of  ice  the  gelatin  is  spread  upon  its  walls  as  in  an  Esmarch  roll 
tube.  For  convenience  in  counting  the  colonies  lines  are  drawn  upon 
the  surface  of  the  cylinder,  dividing  it  into  squares  of  uniform  di- 
mensions. 


GENERAL  RESULTS   OF   RESEARCHES   MADE. 

As  already  stated,  the  presence  of  bacteria  in  the  atmosphere  de- 
pends upon  their  being  wafted  by  currents  of  air  from  surfaces  where 
they  are  present  in  a  desiccated  condition.  That  they  are  not  carried 
away  from  moist  surfaces  is  shown  by  the  fact  that  expired  air  from 
the  human  lungs  does 'not  contain  microorganisms,  although  the  in- 
spired air  may  have  contained  considerable  numbers,  and  there  are 
always  a  vast  number  present  in  the  salivary  secretions.  The  moist 
mucous  membrane  of  the  respiratory  passages  constitutes  a  germ 
trap  which  is  much  more  efficient  than  the  glass  slide  smeared  with 
glycerin  used  in  some  of  the  aeroscopes  heretofore  described,  for  it 
is  a  far  more  extended  surface.  As  a  matter  of  fact,  most  of  the  sus- 
pended particles  in  inspired  air  are  deposited  before  the  current  of 
air  passes  through  the  larynx. 

Air  which  passes  over  large  bodies  of  water  is  also  purified  of  its 
germs  and  other  suspended  particles.  The  researches  of  Fischer 
show  that  at  a  considerable  distance  from  the  land  no  germs  an- 
found  in  the  atmosphere  over  the  ocean,  and  that  it  is  only  upon  ap- 
proaching land  that  their  presence  is  manifested  by  the  development 
of  colonies  upon  properly  exposed  gelatin  plates. 

Uffelmann  found,  in  his  researches,  that  in  the  open  fields  th<i 
number  of  living  germs  in  a  cubic  metre  of  air  averaged  two  hundred 
and  fifty,  on  the  sea  coast  the  average  was  one  hundred,  in  the  court- 
yard of  the  University  of  Rostock  four  hundred  and  fifty.  The  nu  n  i  - 
her  was  materially  reduced  after  a  rainfall  and  increased  when  a 
dry  land  wind  prevailed. 

Frankland  found  that  fewer  germs  were  present  in  the  air  in 
winter  than  in  summer,  and  that  when  the  earth  was  covered  with 
snow  the  number  was  greatly  reduced,  as  also  during  a  light  fall  of 
snow  ;  the  air  of  towns  was  found  to  be  more  rich  in  germs  than  the 


BACTERIA   IN   THE   AIR.  633 

air  of  the  country ;  the  lower  strata  of  the  atmosphere  contained 
more  than  the  air  of  elevated  localities. 

Von  Freudenreich  also  found  that  the  air  of  the  country  contained 
fewer  germs  than  that  of  the  city.  Thus  in  the  city  of  Berne  a  cubic 
metre  of  air  often  contained  as  many  as  two  thousand  four  hundred 
germs,  while  the  maximum  in  country  air  was  three  hundred.  His  re- 
sults corresponded  with  those  of  Miquel  in  showing  that  the  number 
of  atmospheric  organisms  is  greater  in  the  morning  and  the  evening, 
between  the  hours  of  6  and  8,  than  during  the  rest  of  the  day.  Neu- 
mann, whose  researches  were  made  in  the  Moabite  Hospital,  found 
the  greatest  number  of  bacteria  in  the  air  in  the  morning  after  the 
patients  able  to  sit  up  had  left  their  beds  and  the  wards  had  been 
swept.  The  number  of  germs  was  then  from  eighty  to  one  hundred 
and  forty  in  ten  litres  of  air,  while  in  the  evening  the  number  fell  to 
four  to  ten  germs  in  ten  litres. 

Miquel  has  given  the  following  summary  of  results  obtained  in 
his  extended  experiments,  made  in  Paris  during  the  years  1881, 1882, 
and  1883  : 


Average 
a 

for  1880  

Number  of  Germs  in  a  Cubic  Metre  of  Air. 

Air  of  Laboratory, 
Montsouri. 

Air  of  Park,  Mont 
souri. 

215 
348 
550 

71 
62 
51 

"   1881  

"   1882  

Eue  de  Rivoli,  average  for  one  year,  750  ;  summit  of  Pantheon,  28  ; 
Hotel-Dieu,  1880,  average  for  four  months,  male  ward  6,300,  female 
ward  5,120  ;  La  Piete  Hospital,  average  of  fifteen  months,  11,100. 

It  must  be  remembered  that  the  figures  given  relate  both  to  bac- 
teria and  to  the  spores  of  mould  fungi,  and  that  the  latter  are  com- 
monly the  most  numerous  when  the  experiment  is  made  in  the  open 
air.  Petri  has  shown  that  when  gelatin  plates  are  exposed  in  the  air 
the  relative  number  of  spores  of  mould  fungi  deposited  upon  them  is 
less  than  is  obtained  in  aspiration  experiments. 

The  number  of  colonies  which  develop  on  exposed  plates  does  not 
represent  the  full  number  of  bacteria  deposited,  for  these  colonies 
very  frequently  have  their  origin  in  a  dust  particle  to  which  several 
bacteria  are  attached,  or  in  a  little  mass  of  organic  material  contain- 
ing a  considerable  number. 

It  is  generally  conceded  that  sea  air  and  country  air  are  more 
wholesome  than  the  air  of  cities,  and  especially  of  crowded  apart- 
ments, in  which  the  number  of  bacteria  has  been  shown  to  be  very 
much  greater.  But  it  would  be  a  mistake  to  ascribe  the  sanitary 
value  of  sea,  country,  and  mountain  air  to  the  relatively  small  num- 
44 


634  BACTERIA   IN   THE   AIR. 

ber  of  bacteria  present  in  such  air.  There  are  other  important  fac- 
tors to  be  considered,  and  we  have  no  satisfactory  evidence  that  the 
number  of  saprophytic  bacteria  present  in  the  air  has  an  important 
bearing  upon  the  health  of  those  who  respire  ib.  We  do  know  that 
the  confined  air  of  crowded  apartments,  and  especially  of  factories 
in  which  a  large  quantity  of  dust  is  suspended  in  the  air,  predisposes 
those  breathing  such  air  to  pulmonary  diseases  and  lowers  the  gen- 
eral standard  of  health.  But  it  has  not  been  proved  that  this  is  due 
to  the  presence  of  bacteria.  Infectious  diseases  may,  under  certain 
circumstances,  be  communicated  by  way  of  the  respiratory  passages 
as  a  result  of  breathing  air  containing  in  suspension  pathogenic  bac- 
teria ;  but  there  is  reason  to  believe  that  this  occurs  less  frequently 
than  is  generally  supposed. 

Kriiger  has  shown  that  the  dust  of  a  hospital  ward  in  which  pa- 
tients with  pulmonary  consumption  expectorated  occasionally  upon 
the  floor  contained  tubercle  bacilli.  This  was  proved  by  wiping  up 
the  dust  on  a  sterilized  sponge,  washing  this  out  in  bouillon,  and  in- 
jecting this  into  the  cavity  of  the  abdomen  of  guinea-pigs.  Two 
animals  out  of  sixteen  injected  became  tuberculous.  In  pulmonic 
anthrax,  which  occasionally  occurs  in  persons  engaged  in  sorting 
wool — "wool-sorters'  disease" — infection  occurs  as  a  result  of  the 
respiration  of  air  containing  the  spores  of  the  anthrax  bacillus. 

Among  the  non-pathogenic  saprophytes  found  in  the  air  certain 
aerobic  micrococci  appear  to  be  the  most  abundant,  and,  as  a  rule, 
bacilli  are  not  found  in  great  numbers  or  variety.  In  some  localities 
various  species  of  sarcinse  are  especially  abundant.  The  following 
is  a  partial  list  of  the  species  which  have  been  shown  by  the  researches 
of  various  bacteriologists  to  be  occasionally  present  in  the  air.  But, 
as  heretofore  remarked,  their  presence  is  to  be  regarded  as  acci- 
dental, and  so  far  as  we  know  there  is  no  bacterial  flora  properly  be- 
longing to  the  atmosphere  : 

Micrococcus  ureae  (Pasteur),  Diplococcus  rosens  (Burnm),  Diplococcus 
citreus  conglomeratus  (Bumm),  Micrococcus  radiatus  (Fliigge),  Micrococcus 
flavus  desidens  (Fliigge),  Micrococcus  flavus  liquefaciens  (Fliigge),  Micro- 
coccus  tetrageiius  versatilis  (Sternberg),  Micrococcus  pyogenes  aureus  (Rosen- 
bach),  Micrococcus  pyogenes  citreus  (Passet),  Micrococcus  cinnabareus 
(Fliigge),  Micrococcus  flavus  tardigradus  (Fliigge),  Micrococcus  versifolor 
(Fliigge),  Micrococcus  viticulosus  (Katz),  Micrococcus  candidans  (Fliigge), 
Pediococcus  cerevisiae  (Balcke),  Sarcina  lutea  (Schrfiter),  Sarcina  rosea 
(Sckroter),  Sarcina  aurantiaca,  Sarcina  alba,  Sarcina  Candida  (Reinkr). 
Bacillus  tumescens  (Zopf),  Bacillus  subtilis  (Ehrenberg).  Bacillus  multipedi- 
culosus  (Fliigge),  Bacillus  mesentericus  fusous  (Flu<™^),  Bacillus  mesenteri- 
cus  ruber  (G-lobig),  Bacillus  inflates  (A.  Koch),  Bacillus  mesentericus  vul- 
gatus,  Bacillus  prpdigiams,  Bacillus  aerophilus  (Liborius),  Bacillus  pestifer 
(Frankland),  Spirillum  aureum  (Weibel),  Spirillum  flavescens  ( Weibel) ,  Spi- 
rillum flavum  (Weibel),  Bacillus  Havaniensis  (Sternberg). 

In  the  researches  of  Welz,  made  in  the  vicinity  of  Freiburg,  twenty- 
three  different  micrococci  and  twenty-two  bacilli  were  obtained  from  the 


air. 


BACTERIA   IN  THE  AIR.  635 


ADDITIONAL   NOTES   UPON  BACTERIA   IN   THE   AIR. 

Ruete  and  Enoch  (1895)  have  examined  the  air  of  closed  schoolrooms 
with  the  following  results.  Eighteen  different  species  were  obtained,  only 
one  of  which  proved  to  be  pathogenic  for  mice,  guinea-pigs,  and  rabbits.  The 
number  of  bacteria  per  cubic  metre  varied  from  1,500  to  3,000,000,  the  aver- 
age being  about  268, 000.  The  observations  were  made  during  the  winter 
months. 

Marpmann  (1893),  in  his  examination  of  dust  collected  in  the  streets  of  Leip- 
zig for  tubercle  bacilli,  -  obtained  positive  results  from  a  considerable  pro- 
portion of  the  specimens  examined.  Evidently  these  bacilli  in  dust  from  the 
streets  are  liable  to  be  blown  into  the  air  and  deposited  upon  the  mucous 
membrane  of  the  respiratory  passages  of  those  breathing  this  air.  Christian! 
(1893)  has  shown  that,  as  a  rule,  no  bacteria  are  present  in  the  air  at  an  alti- 
tude of  one  thousand  metres  or  more  above  the  soil  (air  collected  during 
balloon  ascensions). 

Dyar  (1895)  has  made  a  careful  study  of  the  microorganisms  found  in  the 
air  in  the  city  of  New  York.  He  has  described  numerous  species  of  micro- 
cocci  and  bacilli  found  chiefly  in  the  air  of  the  hallway  of  the  College  of 
Physicians  and  Surgeons.  Some  of  these  are  new  and  some  have  been 
identified  as  previously  described  species. 


II. 

BACTERIA  IN  WATER. 

THE  water  of  the  ocean,  of  lakes,  ponds,  and  running  streams 
necessarily  contains  bacteria,  as  they  are  constantly  being  carried 
into  it  by  currents  of  air  passing  over  the  neighboring  land  surfaces, 
and  by  rain  water  which  washes  suspended  microorganisms  from 
the  atmosphere ;  and,  as  such  water  contains  more  or  less  organic 
material  in  solution,  many  of  the  saprophytic  bacteria  multiply  in  it 
abundantly.  It  is  only  in  the  water  of  springs  and  wells  which 
comes  from  the  deeper  strata  of  the  soil  that  they  are  absent.  The 
number  and  variety  of  species  present  in  water  from  any  given 
source  will  depend  upon  conditions  relating  to  the  amount  of  organic 
pabulum,  the  temperature,  the  depth  of  the  water,  the  fact  of  its 
being  in  motion  or  at  rest,  its  pollution  from  various  sources,  etc. 
The  comparatively  pure  water  of  lakes  and  running  streams  contains 
a  considerable  number  of  bacteria  which  find  their  normal  habitat 
in  such  waters  and  which  multiply  abundantly  in  them,  notwith- 
standing the  small  quantity  of  organic  matter  and  salts  which  they 
contain.  The  water  of  stagnant,  shallow  pools,  and  of  sluggish 
streams  into  which  sewage  is  discharged,  contains  a  far  greater 
number  and  a  greater  variety  of  species. 

The  study  of  these  bacteria  in  water  has  received  much  attention 
on  account  of  the  sanitary  questions  involved,  relating  to  the  use  of 
water  from  various  sources  for  drinking  purposes.  In  the  present 
section  we  shall  first  give  an  account  of  the  methods  of  bacteriologi- 
cal water  analysis,  and  then  a  condensed  statement  of  results  ob- 
tained in  the  very  numerous  investigations  which  have  been  made. 

A  very  important  point  to  be  kept  in  view  is  the  fact  that  a  great 
increase  in  the  number  of  bacteria  present,  in  samples  of  water  col- 
lected for  investigation,  is  likely  to  occur  if  these  samples  are  kept 
for  some  time.  A  water  which,  for  example,  contains  only  two 
hundred  to  three  hundred  bacteria  per  cubic  centimetre  when  the  ex- 
amination is  made  at  once,  may  contain  several  thousand  at  the  end 
of  twenty-four  hours,  and  at  the  end  of  the  second  or  third  day 
twenty  thousand  or  more  may  be  present  in  the  same  quantity. 


BACTERIA   IN   WATER.  637 

Later,  on  account  of  the  exhaustion  of  organic  pabulum,  the  num- 
ber is  again  reduced  as  the  bacteria  present  gradually  lose  their 
vitality.  Under  these  circumstances  it  is  evident  that  an  estimate  of 
the  number  of  bacteria  present  in  water  from  a  given  source  can 
have  no  value,  unless  a  sample  is  tested  by  bacteriological  methods 
within  a  short  time  after  it  has  been  collected.  Not  more  than  an 
hour  or  two  should  be  allowed  to  elapse,  especially  in  warm  weather. 
By  placing  the  water  upon  ice  the  time  may  be  extended  somewhat, 
but  Wolffhugel  has  shown  that  the  number  of  germs  is  gradually 
diminished  when  water  is  preserved  in  this  way,  and  it  will  be  safest 
to  make  an  immediate  examination  when  this  is  practicable. 

The  collection  may  be  made  in  a  sterilized  Erlenmeyer  flask  pro- 
vided with  a  cotton  air  filter,  or  in  a  bottle  having  a  ground-glass 
stopper  which  has  been  wrapped  in  tissue  paper  and  sterilized  for  an 
hour  or  more  at  150°  C.  in  the  hot-air  oven.  Or  the  small  flasks  with 
a  long  neck  may  be  used,  as  first  recommended  by  Pasteur.  These 
are  prepared  as  follows  :  The  bulb  is  first  gently  heated,  and  the  ex- 
tremity of  the  tube  dipped  into  distilled  water,  which  mounts  into 


FIG.  196. 

the  bulb  as  it  cools ;  the  water  is  then  made  to  boil,  and  when  all 
but  a  drop  or  two  has  escaped  and  the  bulb  is  filled  with  steam  the 
extremity  of  the  tube  is  hermetically  sealed.  When  the  steam  has 
condensed  by  the  cooling  of  the  bulb  a  partial  vacuum  is  formed, 
and  the  tube  is  ready  for  use  at  any  time.  It  is  filled  with  water  by 
breaking  off  the  sealed  extremity  under  the  surface  of  the  water  of 
which  a  sample  is  desired.  This  is  done  with  sterilized  forceps,  and 
care  must  be  taken  that  the  exterior  of  the  tube  is  properly  sterilized 
before  the  collection  is  made.  The  end  is  immediately  sealed  in  the 
flame  of  a  lamp.  A  difficulty  with  these  vacuum  tubes  is  that  they 
are  so  completely  filled  with  water  that  this  cannot  be  readily  drawn 
from  them  again  in  small  quantities.  The  writer  therefore  prefers 
to  make  the  collection  in  a  tube  shaped  as  shown  in  Fig.  196,  in  which 
a  partial  vacuum  is  formed  just  before  the  collection  by  heating  the 
air  in  the  bulb.  The  water  mounts  into  the  tube  as  the  air  in  the 
bulb  cools,  and  is  readily  forced  out  again  for  making  cultures  by 
applying  gentle  heat  to  the  bulb.  As  a  lamp  is  needed  to  seal  the  end 
of  the  tube  in  either  case,  there  is  no  special  advantage  in  having  a 
vacuum  formed  in  advance,  and,  as  stated,  the  vacuum  tubes  are  so 


638 


BACTERIA    IN  WATER. 


nearly  filled  with  water  that  it  is  not  so  simple  a  matter  to  obtain  the 
contents  for  our  culture  experiments  without  undue  exposure  to  at- 
mospheric germs.  In  practice  small  glass  bottles  with  ground-glass 
stoppers  will  be  found  most  convenient,  and,  when  properly  steril- 
ized, are  unobjectionable.  They  should  be  filled  at  a  little  distance 
below  the  surface,  as  there  is  often  a  deposit  of  dust  upon  the  surface 

of  standing  water,  and  sometimes  a 
delicate  film  made  up  of  aerobic  bac- 
teria. When  water  is  to  be  obtained 
from  a  pump  or  a  hydrant  it  should 
be  allowedrto  flow  for  some  time  before 
the  collection  is  made.  To  collect 
water  at  various  depths  the  apparatus 
shown  in  Fig.  197  is  recommended  by 
Lepsius.  An  iron  frame  supports  an 
inverted  flask,  A,  filled  with  sterilized 
mercury  and  containing  about  three 
hundred  cubic  centimetres.  The  flask 
B  is  intended  to  receive  the  mercury 
when,  at  the  desired  depth,  it  is  al- 
lowed to  flow  through  the  capillary 
tube  b.  This  is  sealed  at  the  extremity 
and  bent  as  shown  in  the  figure.  By 
pulling  upon  the  cord  c  this  tube  is 
broken,  and  as  the  mercury  flows  from 
the  flask  this  is  filled  with  water 
through  the  tube  a.  The  extremity 
of  the  broken  tube  b  is  closed  by  the 
mercury  in  the  flask  B  when  A  is  full 
of  water,  and  the  apparatus  can  be 
brought  to  the  surface  with  only  such 
water  as  was  collected  at  the  depth 
from  which  a  sample  was  desired. 

The  bacteriological  analysis  is 
made  by  adding  a  definite  quantity 
of  the  water  under  investigation  to 
liquefied  gelatin  or  agar-gelatin,  and 
making  a  plate  or  Esmarch  roll  tube,  which  is  put  aside  for  the  devel- 
opment of  colonies.  Miquel  and  others  have  preferred  to  use  liquid 
cultures  and  the  method  of  fractional  cultivation  described  in  the 
previous  section.  The  use  of  a  solid  culture  medium  has,  however, 
such  obvious  advantages  that  we  do  not  consider  it  necessary  to  do 
more  than  refer  to  the  other  method  as  one  which,  when  applied 
with  skill  and  patience,  may  give  sufficiently  accurate  results. 


FIG. 


BACTERIA   IX    WATER.  639 

The  amount  of  water  which  should  be  added  to  the  usual  quan- 
tity of  liquefied  flesh-peptone-gelatin  in  a  test  tube,  in  order  that  the 
colonies  which  develop  may  be  well  separated  from  each  other  and 
easily  counted,  can  only  be  determined  by  experiment.  If  the  water 
is  from  an  impure  source  a  single  drop  may  be  too  much,  and  it  will 
be  necessary  to  dilute  it  with  distilled  water  recently  sterilized.  But 
for  .ordinary  potable  water  it  will  usually  be  best,  in  a  first  experi- 
ment, to  make  two  trials,  one  with  one  cubic  centimetre  and  one 
with  one-half  cubic  centimetre  added  to  the  liquefied  nutrient  gelatin. 
The  water  in  the  collecting  bottle  should  be  shaken,  to  distribute  the 
bacteria  which  may  have  settled  to  the  bottom,  before  drawing  off  by 
means  of  a  sterilized  pipette  the  amount  used  for  the  experiment,  and 
the  germs  present  in  it  are  to  be  distributed  through  the  liquefied 
gelatin  by  gently  moving  the  tube  to  and  fro. 

Koch's  method  of  preparing  a  gelatin  plate  is  illustrated  in  Fig. 
198.  A  glass  dish,  containing  ice  water  and  covered  with  a  large 


FIG.  198. 

plate  of  glass,  is  supported  upon  a  levelling  tripod.  By  means  of  a 
spirit  level  this  is  adjusted  to  a  horizontal  position,  so  that  when  the 
liquefied  gelatin  is  poured  upon  the  smaller  sterilized  glass  plate,  seen 
in  the  centre  of  the  large  plate  of  glass,  it  will  not  flow,  but  may  be 
evenly  distributed  over  the  surface  by  means  of  a  sterilized  glass  rod. 
The  glass  cover  resting  against  the  side  of  the  apparatus  is  placed 
over  the  gelatin  plate  while  it  is  cooling,  to  protect  it  from  atmo- 
spheric germs,  and  when  the  gelatin  is  hard  the  plate  is  transferred 
to  a  shallow  glass  dish,  which  is  kept  at  a  temperature  of  about 
20°  C.  for  several  days  for  the  development  of  colonies.  It  is  difficult 
to  count  colonies  when  more  than  five  thousand  develop  upon  a  plate 
of  the  usual  size,  and  for  this  reason  it  will  be  best  to  repeat  the  ex- 
periment with  a  smaller  quantity  of  water  from  the  same  source,  if 
this  is  at  hand,  rather  than  to  attempt  to  count  an  overcrowded 
plate.  Before  pouring  the  gelatin  upon  the  plate  the  lip  of  the  test 
tube  containing  it  should  be  sterilized  by  passing  it  through  a  flame,- 
The  liquefied  gelatin  should  be  carefully  distributed  to  cover  a  rect- 


640  BACTERIA   IN  WATER. 

angular  surface  and  leaving  a  margin  of  about  one  centimetre  around 
the  edge  of  the  plate.  The  Koch's  dish  in  which  the  gelatin  plate  is 
placed  for  the  development  of  colonies  should  be  carefully  sterilized 
by  heat  or  by  washing  it  out  with  a  sublimate  solution.  A  circular 
piece  of  filtering  paper,  saturated  with  sublimate  solution  or  distilled 
water,  is  placed  at  the  bottom  of  the  lower  dish  to  keep  the  air  in  a 
moist  condition  and  prevent  drying  of  the  gelatin.  Usually  two  or 
three  plates  made  at  the  same  time  are  placed  one  above  the  other  on 
glass  supports  made  for  this  purpose.  If  many  liquefying  organisms 
are  present  it  will  be  necessary  to  count  the  colonies  before  these  run 
together — usually  on  the  second  day  ;  but  in  the  absence  of  liquefy- 
ing colonies  it  is  best  to  wait  until  the  third,  or  even  the  fifth  day,  as 
the  number  of  visible  colonies  and  the  ease  of  counting  them  will  be 
greater  than  at  an  earlier  date.  The  development  of  a  few  scattered 
liquefying  colonies  which  threaten  to  spoil  the  plate  may  be  arrested 
by  taking  up  the  liquefied  gelatin  from  each  with  a  bit  of  filtering 
paper,  and  then,  by  means  of  a  camel's-hair  brush,  applying  a  solu- 
tion of  potassium  permanganate  to  the  margin  of  the  colony.  The 
growth  of  colonies  of  mould  fungi,  which  have  developed  from  spores 
from  the  atmosphere  falling  upon  the  plate  while  it  is  exposed,  can 
be  checked  by  the  application  of  collodion  containing  bichloride  of 
mercury. 

Counting  of  the  colonies  is  a  simple  matter  when  they  are  few 
in  number ;  when  they  are  numerous  it  is  customary  to  place  the 
plate  over  a  dark  background,  and  to  place  above  it  a  glass  plate 
divided  into  square  centimetres  by  lines  ruled  with  a  diamond.  By 
means  of  a  lens  of  low  power  the  colonies  in  a  certain  number  of 
squares  are  counted  and  the  average  taken.  This  multiplied  by  the 
number  of  square  centimetres  in  the  gelatin-covered  surface  gives 
approximately  the  entire  number  of  colonies  which  have  developed 
from  the  amount  of  water  used  in  the  experiment. 

Instead  of  using  Koch's  original  plate  method,  as  above  described, 
the  shallow,  covered  glass  dishes  recommended  by  Petri  may  be 
employed.  These  are  from  one  to  one  and  one-half  centimetres  high 
and  from  ten  to  fifteen  centimetres  in  diameter.  The  liquefied  gel- 
atin is  poured  into  the  lower  dish  and  the  cover  at  once  placed  over 
it.  The  gelatin  does  not  dry  out  very  soon,  but,  if  necessary,  several 
of  these  Petri's  dishes  may  be  placed  in  a  larger  jar,  which  serves  as 
a  moist  chamber. 

The  roll  tubes  of  Esmarch  may  also  be  used,  and  have  the  ad- 
vantage that  accidental  colonies  from  air-borne  germs  are  excluded. 
The  counting  of  colonies  is  not  quite  as  easy,  but  by  the  use  of  a 
mounted  lens  especially  designed  for  the  purpose  it  is  attended  with 
no  great  difficulty.  The  surface  of  the  tube  is  divided  into  squares 


BACTERIA   IN   WATER.  641 

by  colored  lines,  and  the  number  of  colonies  in  several  squares  is 
counted  in  order  to  obtain  an  average  and  estimate  the  entire 
number. 

Water  which  contains  numerous  liquefying  bacteria  had  better 
be  examined  by  the  use  of  nutrient  agar  instead  of  gelatin;  and  in 
very  warm  weather  it  will  be  necessary  to  use  an  agar  medium,  as 
ten-per-cent  gelatin  is  likely  to  melt  if  the  temperature  goes  above 
22°  C.  A  difficulty  in  the  use  of  agar  for  plates  consists  in  the  lia- 
bility of  the  film  to  slip  from  the  glass.  This  may  be  remedied  to 
some  extent  by  adding  a  few  drops  of  a  concentrated  solution  of  gum 
acacia  to  the  liquefied  agar  medium.  Petri's  dishes  are  well  adapted 
for  the  use  of  the  agar  medium,  as  the  objection  referred  to  does  not 
apply  to  them.  The  gelatin-agar  medium,  containing  5  per  cent 
of  gelatin  and  0. 75  per  cent  of  agar,  may  also  be  used  with  advan- 
tage in  the  bacteriological  analysis  of  water.  Much  stress  was  at 
one  time  laid  upon  the  enumeration  of  liquefying  colonies,  upon 
the  supposition  that  the  liquefying  bacteria  were  especially  harmful 
as  compared  with  the  non-liquefying,  and  that  a  water  containing 
many  liquefying  colonies  was  to  be  looked  upon  with  suspicion.  We 
now  know,  however,  that  there  are  many  common  and  harmless 
saprophytes  which  cause  the  liquefaction  of  gelatin,  and  that  some 
of  the  most  dangerous  pathogenic  bacteria  do  not  liquefy  gelatin. 
This  distinction  has  therefore  no  special  value,  and  the  question  for 
bacteriologists  to-day  is  not  how  large  is  the  comparative  number  of 
liquefying  colonies,  but  what  species  are  represented  by  the  colonies 
present,  liquefying  and  non-liquefying,  and  what  are  the  special 
pathogenic  properties  of  each.  The  answer  to  these  questions,  in 
the  case  of  any  particular  water  supply,  calls  for  special  knowledge 
and  great  patience  and  care  in  the  isolation  in  pure  cultures,  and 
careful  study  of  the  various  species  present. 

It  is  now  generally  recognized  that  a  mere  enumeration  of  the 
number  of  colonies  which  develop  from  a  water  under  investigation 
is  not  a  sufficient  indication  upon  which  to  found  an  opinion  as  to  its 
potability.  An  excessive  number  of  bacteria  is  an  indication  that 
the  water  contains  a  large  amount  of  the  organic  material  which 
serves  as  pabulum  for  these  microorganisms.  But  the  chemists  are 
able  to  determine  the  amount  of  organic  matter  present  in  water 
with  greater  precision  ;  and,  as  we  have  seen,  the  number  of  bacteria 
may  increase  many-fold  in  water  which  is  kept  standing  in  the  labo- 
ratory for  two  or  three  days  in  a  well-corked  bottle.  As  a  matter  of 
fact,  the  enumeration  of  bacteria  in  water,  although  it  has  given  us 
results  of  scientific  interest,  has  not  materially  added  to  the  methods 
previously  applied  for  estimating  the  sanitary  value  of  water  ob- 
tained from  various  sources  for  drinking  purposes.  But  the  bacte- 


642  BACTERIA   IX   WATER. 

riological  examination  may  prove  to  be  of  great  value  if  it  succeeds 
in  demonstrating  the  presence  of  certain  pathogenic  bacteria  and  in 
thus  preventing  the  use  of  a  dangerous  water.  We  do  not  mean  to 
say,  however,  that  an  enumeration  of  the  bacteria  present  in  drink- 
ing water  has  no  practical  value.  An  excessive  number  indicates  an 
excessive  amount  of  organic  pabulum,  which  may  have  come  from 
a  dangerous  source;  and  the  dangerous  pathogenic  bacteria  are  not 
only  more  likely  to  be  present  in  such  water,  but  they  can  more 
readily  multiply  in  it,  while  in  a  pure  water  they  would  fail  to  in- 
crease in  number,  and,  as  has  been  shown  by  experiment,  would  die 
out  within  a  short  time. 

The  number  of  bacteria  present  in  rain  water,  or  in  snow  which 
has  recently  fallen,  varies  greatly  at  different  times.  Naturally  the 
number  is  greater  when  the  surface  of  the  earth  is  dry  and  the  at- 
mosphere loaded  with  dust  by  currents  of  wind  passing  over  it,  and 
less  when  the  surface  is  moist  and  the  atmosphere  has  been  purified 
by  recent  rains. 

In  snow  from  the  surface  of  a  glacier  in  Norway,  Schmelck  found 
two  bacteria  and  two  spores  of  mould  fungi  per  cubic  centimetre  of 
water  from  the  melted  snow.  Ganowski,  in  experiments  made  with 
freshly  fallen  snow  collected  in  the  vicinity  of  Kiew,  obtained  the  fol- 
lowing results  :  February  3d,  1888  :  temperature  of  the  air,  7.2°  C.; 
snowfall,  0. 1  millimetre ;  number  of  bacteria  in  1  cubic  centimetre 
of  water  from  melted  snow,  34  in  one  sample  and  38  in  another. 
February  20th,  1888:  temperature,  11.1°  C.  ;  snowfall,  1.1  milli- 
metres ;  number  of  bacteria  in  one  sample,  203,  in  another  384. 

Miquel  obtained  from  rain  water  collected  at  Montsouri  during  a 
rainy  season  4. 3  germs  per  cubic  centimetre  ;  in  rain  water  collected 
in  the  centre  of  the  city  of  Paris,  19  per  cubic  centimetre. 

Hailhaa  also  been  shown  to  contain  bacteria  inconsiderable  num- 
bers. Bujwid  found  in  hailstones  which  fell  at  Warsaw  21,000 
bacteria  in  1  cubic  centimetre  ;  but  this  is  exceptional,  and  is  supposed 
to  be  due  to  the  fact  that  surface  water  had  been  carried  into  the  air 
by  the  storm  and  frozen.  Fontin  examined  hail  which  fell  in  St. 
Petersburg,  and  obtained  an  average  of  729  bacteria  per  cubic  centi- 
metre of  water  from  the  melted  hail. 

River  water  has  been  carefully  examined  by  numerous  bacterio- 
logists in  various  localities  and  at  different  seasons  of  the  year.  We 
give  below  some  of  the  results  reported  : 

Water  of  the  Seine  at  Choisy,  before  reaching  Paris,  300  ;  at 
Bercy,  1,200 ;  at  Saint-Denis,  after  receiving  the  sewer  water  from 
the  city,  200,000  germs  per  cubic  centimetre  (Miquel). 

Water  of  the  Spree  beyond  Kopenick,  82,000  ;  two  hundred  steps 
below  the  mouth  of  the  Wuhle,  118,000  ;  ill  Berlin  above  the  mouth 


BACTERIA  IN   WATER.  643 

of  the  Panke,   940,000;  below  the  mouth  of  the  Panke,   1,800,000 
(Koch). 

Water  of  the  Main  above  the  city  of  Wiirzburg,  in  the  month  of 
February,  520  ;  below  the  city,  15,500  (Rosenberg). 

Water  of  the  Potomac,  at  Washington,  in  1886  :  January,  3,774  ; 
February,  2,536;  March,  1,210;  April,  1,521;  May,  1,064;  June, 
348  ;  July,  255  ;  August,  254 ;  September,  178 ;  October,  75  ;  No- 
vember, 116  ;  December,  967  (Theobald  Smith). 

The  Thames,  in  the  autumn  of  1885,  in  the  vicinity  of  London 
Bridge  two  hours  after  high  water,  contained  45,000  germs  per  cubic 
centimetre ;  the  water  of  the  Lea  at  Lea  Bridge,  4,200,000  (Bisch- 
off). 

The  Neva  inside  the  city  of  St.  Petersburg,  in  September,  1883, 
contained  1,500  in  one  sample  and  1,040  in  another  ;  in  November 
(20th),  6,500  (Poehl). 

The  water  of  the  Oder,  collected  within  the  limits  of  the  city  of 
Stettin,  was  found  by  Link  to  contain  from  5,240  to  15,000  bacteria 
per  cubic  centimetre  ;  that  of  the  Limmat,  at  Zurich,  346  in  one 
specimen  and  508  in  another  (Cramer). 

Lake  water,  as  a  rule,  contains  fewer  bacteria  than  river  water. 

Wolffhiigel,  in  researches  extending  from  July,  1884,  to  July, 
1885,  obtained  from  the  water  of  the  Tegeler  Lake  an  average  of  396 
bacteria  per  cubic  centimetre.  Cramer  obtained  an  average  of  168 
per  cubic  centimetre  during  the  months  of  October,  December,  and 
January,  1884,  from  the  water  of  Lake  Zurich  ;  in  June  of  the  same 
year  the  average  of  42  examinations  gave  71  per  cubic  centimetre. 
In  Lake  Geneva,  Fol  and  Dunant  obtained  from  water  collected  some 
distance  from  the  shore  an  average  of  38  bacteria  per  cubic  centi- 
metre. 

Ice  which  is  usually  collected  from  lakes  and  rivers  contains  a 
greater  or  less  number  of  bacteria,  according  to  the  depth  and  purity 
of  the  water.  The  ice  used  in  Berlin,  collected  from  the  surface  of 
lakes  and  rivers  in  the  vicinity  of  the  city,  contains  from  a  few  hun- 
dred to  25,000  bacteria  to  the  cubic  centimetre  (Friinkel).  In  the  ex- 
periments of  Heyroth  samples  of  ice  from  the  same  source  gave  less 
than  100  per  cubic  centimetre  in  three,  from  100  to  500  in  eight,  from 
500  to  1,000  in  six,  from  1,000  to  5,000  in  seven,  and  14,400  in  one. 

Prudden  obtained  from  Hudson  River  ice,  put  up  six  miles  below 
the  city  of  Albany,  an  average  of  398  bacteria  per  cubic  centimetre 
from  transparent  ice,  and  in  the  superficial  "snow  ice"  9,187.  Ice 
collected  lower  down  the  river  contained  an  average  of  189  in  the 
transparent  and  3,693  in  the  snow  ice. 

Ice  from  the  Dora  at  Turin  was  found  by  Bordoni-Uffreduzzi  to 
contain  from  120  to  3,546  bacteria  per  cubic  centimetre. 


644  BACTERIA   IX   WATER. 

Hydrant  water,  as  supplied  to  cities,  has  received  the  attention 
of  numerous  investigators.  The  water  supply  of  Berlin  was  ex- 
amined by  Plagge  and  Proskauer  at  intervals  of  a  week  from  June, 
1885,  to  April,  1886.  Their  tabulated  results  show  considerable 
variations.  We  give  the  figures  for  a  single  day,  June  30th,  1885  : 
Stralauer  works,  water  of  the  Spree,  unfiltered  4,400,  filtered  53  ; 
Tegeler  works,  water  of  the  lake,  unfiltered  880,  filtered  44  ;  high  re- 
servoir at  Charlottenberg,  71  ;  75  W.  Wilhelmstrasse,  121  ;  Fried- 
richstrasse,  41-42  S.  W.,  160 ;  Schmidstrasse,  165  E.,  51  ;  Friedrich- 
strasse,  126  K,  151 ;  Weinmeisterstrasse,  15  C.,  63. 

Wells  which  are  supplied  by  water  from  deep  strata  contain  few 
bacteria,  unless  contaminated  by  surface  water  in  which  they  are 
usually  very  abundant.  Roth  examined  the  water  of  sixteen  surface 
wells  in  Belgard,  which  has  a  very  porous  subsoil,  and  found  from 
4,500  to  5,000  bacteria  in  three,  from  7,800  to  15,000  in  six,  from 
18,000  to  35,000  in  six,  and  130,000  per  cubic  centimetre  in  one. 

Forty-seven  wells  in  Stettin,  the  water  of  which  was  examined  by 
Link,  gave  the  following  results  :  Less  than  100  in  six,  100  to  500  in 
twenty-one,  and  in  the  remainder  (sixteen)  from  1,000  to  18,000. 

Sixty-four  wells  in  Mainz  examined  by  Egger,  and  53  in  Gotha 
by  Becker,  gave  more  favorable  results  ;  the  number  of  wells  in  the 
former  city,  in  which  less  than  100  colonies  developed  from  1  cubic 
centimetre,  was  34,  and  in  the  latter  the  same  (34).  Bolton  examined 
the  water  of  13  wells  in  Gottingen,  and  found  but  1  in  which  the 
number  of  colonies  from  1  cubic  centimetre  was  less  than  100  ;  in  12 
the  number  varied  from  180  to  4,940. 

The  water  of  deep  wells  and  springs  may  be  entirely  free  from 
bacteria,  or  nearly  so.  Egger  found  in  the  water  of  an  artesian  well 
at  Mainz  4  bacteria  per  cubic  centimetre,  and  the  same  number  was 
found  by  Hueppe  in  the  deep  well  at  the  Wiesbaden  slaughter-house. 
The  artesian  well  at  the  gasworks  of  Kiel  was  found  by  Brennig  to 
contain  from  6  to  30  bacteria  per  cubic  centimetre.  In  a  spring  at 
Batiolettes,  Fol  and  Dunant  found  57  bacteria  per  cubic  centimetre. 
Fiirbringer  obtained  from  springs  at  Jena  156  from  one,  51  from 
another,  32  from  another,  and  109  from  another.  The  water  supplied 
to  Danzig  from  the  Prangenaur  Spring  was  found  in  several  experi- 
ments to  be  free  from  bacteria  (Freimuth). 

In  a  summary  of  results  obtained  in  various  German  cities  Tie* 
mann  and  Gartner  find  that  sixty-nine  per  cent  of  the  wells  from 
which  samples  of  water  were  examined  contained  less  than  500  bac- 
teria per  cubic  centimetre. 

The  water  of  seivers  is  naturally  rich  in  bacteria.  Miquel  found 
that  at  Clichy  the  sewer  water  contained  6,000,000  bacteria  per  cubic 
centimetre.  Bischoff  found  in  water  from  London  sewers  7,500,000, 


BACTERIA   IN  WATER.  645 

and  numerous  observations  show  that  the  number  of  bacteria  in  river 
water  is  greatly  increased  in  the  vicinity  of  and  below  the  mouths 
of  city  sewers. 

We  conclude  from  the  experimental  data  recorded  that  water 
containing  less  than  100  bacteria  to  the  cubic  centimetre  is  presum- 
ably from  a  deep  source  and  uncontaminated  by  surface  drainage, 
and  that  it  will  usually  be  safe  to  recommend  such  water  for  drink- 
ing purposes,  unless  it  contains  injurious  mineral  substances. 
Water  that  contains  more  than  500  bacteria  to  the  cubic  centimetre, 
although  it  may  in  many  cases  be  harmless,  is  to  be  looked  upon 
with  some  suspicion,  and  water  containing  1,000  or  more  bacteria  is 
presumably  contaminated  by  sewage  or  surface  drainage  and  should 
be  rejected  or  filtered  before  it  is  used  for  drinking  purposes.  But, 
as  heretofore  stated,  the  danger  does  not  depend  directly  upon  the 
number  of  bacteria  present,  but  upon  contamination  with  pathogenic 
species  which  are  liable  to  be  present  in  surface  water  and  sewage. 
In  swallowing  a  glassful  of  pure  spring  water  a  number  of  bacteria 
from  the  buccal  cavity  are  washed  away  and  carried  into  the  stomach, 
which,  if  enumerated,  would  doubtless  far  exceed  in  numbers  those 
found  in  the  most  impure  river  water. 

The  number  of  bacteria  does  not  depend  alone  upon  the  amount 
of  organic  pabulum  contained  in  a  water,  and  cannot  be  depended 
upon  in  forming  an  estimate  of  this ;  for,  as  has  been  shown  by 
Bolton,  certain  water  bacteria  multiply  abundantly  in  water  con- 
taining comparatively  little  organic  matter,  while  other  species  fail 
to  grow  unless  the  quantity  is  greater.  In  a  water  containing  con- 
siderable nutrient  material  the  water  bacteria  may  be  restrained  in 
their  development  by  other  species  present  until  the  amount  of  pabu- 
lum is  reduced  so  that  these  no  longer  thrive,  when  the  common 
water  bacteria  will  take  the  precedence,  and  an  enumeration  may 
show  a  greater  number  of  colonies  than  at  first.  But,  in  general, 
water  rich  in  organic  material  contains  a  greater  number  of  bacteria 
and  a  greater  variety  of  species  than  that  which  is  comparatively 
pure. 

That  certain  bacteria  may  multiply  in  water  which  has  been 
carefully  distilled  has  been  shown  by  Bolton  and  others.  Two  com- 
mon water  bacteria — Micrococcus  aquatilis  and  Bacillus  erythrospo- 
rus — multiplied  abundantly  in  doubly  distilled  water,  and  when 
this  water  was  again  sterilized  and  re-inoculated  with  one  of  these 
species  the  same  abundant  increase  occurred.  This  was  repeated  six 
times  with  the  same  result  (Bolton).  Computing  the  number  of 
these  water  bacteria  in  ten  cubic  centimetres  of  distilled  water  at 
twenty  millions,  and  estimating  their  specific  gravity  at  one,  and  the 
diameter  of  the  individual  cells  at  one  /',  the  total  weight  of  the  entire 


646  BACTERIA   IN  WATER. 

number,  according  to  Bolton,  would  be  less  than  one-hundredth 
of  a  milligramme,  and  at  least  three-fourths  of  this  must  consist  of 
water.  The  organic  material  represented  by  this  number  of  bacteria 
would  therefore  be  so  minute  that  it  might  be  supplied  by  dust  par- 
ticles accidentally  falling  into  the  distilled  water. 

Rosenberg  has  shown  that  while  many  of  the  species  which  he 
obtained  in  pure  cultures  from  the  water  of  the  river  Main  multiplied 
in  sterilized  distilled  water,  other  species  quickly  died  out  in  such 
water.  The  growth  of  certain  bacteria  depends  not  only  upon  the 
quantity  of  nutritive  material  present,  but  upon  its  quality,  the  con- 
ditions in  this  regard  being  widely  different  for  different  species. 

In  view  of  the  facts  heretofore  stated  bacteriologists  are  now  giv- 
ing more  attention  to  a  careful  study  of  the  kinds  of  bacteria  pre- 
sent in  their  examinations  of  water.  Rosenberg,  in  his  examinations 
of  the  water  of  the  Main  in  the  vicinity  of  Wiirzburg  (1886),  found 
that  before  the  river  reached  the  city  the  water  contained  more 
micrococci  than  bacilli,  but  that  after  receiving  the  sewage  of  the 
city  the  number  of  bacilli  was  greatly  in  excess. 

Adametz  (1888)  has  described  eighty-seven  species  obtained  by 
him  from  water  in  the  vicinity  of  Vienna  ;  Maschek  found  fifty-five 
different  species  in  the  drinking  water  used  at  Laitmeritz;  and  Tils 
(1890)  has  described  fifty-nine  species  obtained  by  him  from  the  city 
water  supply  at  Freiburg. 

Among  the  pathogenic  bacteria  which  are  liable  to  find  their 
way  into  water  used  for  drinking  purposes,  the  most  important,  from 
a  sanitary  point  of  view,  are  the  bacillus  of  typhoid  fever  and  the 
spirillum  of  Asiatic  cholera.  Both  of  these  microorganisms  are  pre- 
sent in  great  numbers  in  the  excreta  of  persons  suffering  from  the 
specific  forms  of  disease  to  which  they  give  rise,  and  are  consequently 
liable  to  contaminate  wells  and  streams  which  receive  surface  water, 
when  such  excreta  are  thrown  upon  the  surface  or  into  sewers,  etc. 
Epidemics  of  these  diseases  have  frequently  been  traced  to  the  use 
of  such  contaminated  water,  and  in  a  few  instances  the  presence  of 
these  specific  disease  germs  in  water  has  been  demonstrated  by  bac- 
teriological methods.  Laboratory  experiments  indicate,  however, 
that  an  increase  of  these  pathogenic  bacteria  in  drinking  water  is  not 
likely  to  occur,  except  under  special  conditions,  and  that  they  die 
out  after  a  time,  being  at  a  disadvantage  in  the  struggle  for  exist- 
ence constantly  going  on  among  the  numerous  species  which  have 
their  normal  habitat  in  water. 

Bolton,  Frankland,  and  others  have  shown  that  the  anthrax  ba- 
cillus, not  containing  spores,  dies  out  in  hydrant  water  within  five  or 
six  days.  In  the  experiments  of  Kraus  the  anthrax  bacillus  added 
to  well  water,  not  sterilized,  at  a  temperature  of  10.5°  C.,  was  still 


BACTERIA   IN   WATER.  647 

present  in  a  living  condition  on  the  second  day,  but  no  colonies  de- 
veloped after  the  third  day ;  the  typhoid  bacillus  died  out  between 
the  fifth  and  seventh  days  ;  the  cholera  spirillum  was  no  longer  found 
on  the  second  day.  In  the  meantime  the  common  water  bacteria 
had  increased  in  numbers  enormously.  Similar  results  have  been 
reported  by  Hochstetter  and  others.  Hueppe,  in  ten  experiments  in 
which  the  typhoid  bacillus  was  added  to  well  water  of  a  bad  quality, 
found  that  in  two  no  development  of  this  bacillus  occurred  after  the 
fifth  day,  while  a  few  colonies  developed  in  the  other  experiments  as 
late  as  the  tenth  day.  In  these  experiments  the  temperature  was 
comparatively  low  (10.5°  0.).  At  a  higher  temperature  the  experi- 
ments of  Wolffhugel  and  Biedel  show  that  an  increase  may  take 
place.  At  the  room  temperature  (about  20°  C. )  the  typhoid  bacillus 
added  to  distilled  water,  to  well  water,  and  to  Berlin  hydrant  water 
was  still  present,  in  some  instances,  at  the  end  of  thirty-two  days. 
And  it  was  found  that  in  some  cases  a  decrease  in  the  number 
occurred,  then  a  notable  increase,  and  finally  a  second  diminution. 

Koch  found  the  cholera  spirillum  in  a  water  tank  at  Calcutta 
during  a  period  of  fourteen  days,  and  in  his  experiments  showed  that 
it  preserved  its  vitality  in  well  water  for  thirty  days,  in  Berlin  sewer 
water  for  six  to  seven  days,  and  in  the  same  mixed  with  faeces  for 
twenty-seven  hours  only.  In  the  experiments  of  Nicati  and  Rietsch 
the  cholera  spirillum  preserved  its  vitality  in  distilled  water  for 
twenty  days,  in  sewer  water  (of  Marseilles)  thirty-eight  days,  in 
water  of  the  harbor  for  eighty-one  days.  The  numerous  experiments 
recorded  by  the  observers  named,  and  by  Bolton,  Hueppe,  Hoch- 
stetter, Maschek,  Kraus,  and  others,  show  that  while  the  cholera 
spirillum  may  sometimes  quickly  die  out  in  distilled  water,  in ,  other 
experiments  it  preserves  its  vitality  for  several  weeks  (Maschek),  and 
that  it  lives  still  longer  in  water  of  bad  quality,  such  as  is  found  in 
sewers,  harbors,  etc.  Bolton  found  that  for  its  multiplication  a 
water  should  contain  at  least  40  parts  in  100,000  of  organic  material, 
while  the  typhoid  bacillus  grew  when  the  proportion  was  considerably 
less  than  this — 6.7  parts  in  100,000. 

Russell  (1891)  has  studied  the  bacterial  flora  of  the  Gulf  of 
Naples,  and  of  the  mud  at  the  bottom  of  this  gulf,  collected  at 
various  depths  up  to  eleven  hundred  metres.  His  investigations 
show  that  sea  water  does  not  contain  as  many  bacteria  as  an 
equal  volume  of  fresh  water;  that  bacteria  are  found  in  about 
equal  numbers  in  water  from  the  surface  and  in  that  from  various 
depths  ;  that  the  mud  at  the  bottom  constantly  contains  large  num- 
bers of  bacteria ;  that  some  of  the  species  isolated  grow  best  in  a 
culture  medium  containing  sea  water. 

At  a  depth  of  50  metres  the  water  contained  121  bacteria  per  cubic 


648  BACTERIA    IX    WATER. 

centimetre,  and  the  mud  from  the  bottom  245,000  ;  at  100  metres  the 
water  contained  10  and  the  mud  200,000  per  cubic  centimetre ;  at 
500  metres  the  water  contained  22  and  the  mud  12,500  per  cubic 
centimetre  ;  at  1,100  metres  the  mud  contained  24,000. 

The  following  new  species  were  obtained  by  Russell  from  the 
source  mentioned :  Bacillus  thalassophilus,  Cladothrix  intricata, 
Bacillus  granulosus,  Bacillus  limosus,  Spirillum  marinum,  Bacillus 
litoralis,  Bacillus  halophilus. 

The  bacterial  flora  of  fresh  and  sea  water  is  very  extensive,  as 
will  be  seen  by  the  following  list  of  species  which  have  been  described 
by  various  bacteriologists  who  have  given  their  attention  to  its 
study  : 

NON-PATHOGENIC   MICROCOCCI. 

Micrococcus  aurantiacus  (Colin),  Micrococcus  luteus  (Colin),  Micrococcus 
violaceus  (Conn),  Micrococcus  flavus  liquefaciens  (Fliigge),  Micrococcus  fla- 
vus  desidens  (Fliigge),  Micrococcus  radiatus  (Fliigge),  Micrococcus  cinnaba- 
reus  (Fliigge),  Micrococcus  flavus  tardigradus  (Fliigge),  Micrococcus  versi- 
color  (Fliigge),  Micrococcus  agilis  (Ali-Cohen),  Micrococcus fuscus  (Maschek), 
Diplococcus  luteus  (Adametz),  Pediococcus  albus  (Lindner),  Micrococcus 
cerasinus  siccus  (List),  Micrococcus  citreus  (List),  Micrococcus  aquatilis 
(Bolton),  Micrococcus  fervidosus  (Adametz),  Micrococcus  plumosus  (Brauti- 
gam),  Micrococcus  viticulosus  (Katz),  Micrococcus  cremoides  (Zimmermann), 
Micrococcus  carneus  (Zimmermann),  Micrococcus  concentricus  (Zimmer- 
mann), Micrococcus  rosettaceus  (Zimmermann),  Micrococcus ureae  (Pasteur), 
Weisser  Streptococcus  (Maschek),  Wurmformiger  Streptococcus  (Maschek), 
Micrococcus  aerogenes  (Miller),  Sarcina  alba,  Sarcina  Candida  (Reinke), 
Sarcina  lutea. 

PATHOGENIC   MICROCOCCI. 

Staphylococcus  pyogenes  aureus  (Rosenbach),  Micrococcus  of  Heyden- 
reich — "  Micrococcus  Biskra." 

NON-PATHOGENIC  BACILLI. 

Bacillus  arborescens  (Frankland),  Bacillus  viscosus  (Frankland),  Bacil- 
lus aquatilis  (Frankland),  Bacillus  liquidus  (Frankland),  Bacillus  nubilis 
(Frankland),  Bacillus  vermicularis  (Frankland),  Bacillus  aurantiacus 
(Frankland),  Bacillus  coeruleus  (Smith),  Bacillus  Caucus  (Maschek),  Bacil- 
lus albus  putidus  (Maschek),  Bacillus  fluorescens  liquefaciens,  Bacillus  fluo- 
rescens  nivalis  (Schmolck),  Bacillus  lividus  (Plagge  and  Proskauer),  Bacil- 
lus rubidus  (Eisenberg),  Bacillus  sulfureum  (Holschewnikoff),  Bacillus 
violaceus,  Bacillus  gasoformans  (Eisenberg),  Bacillus  liquefaciens  (Eism- 
berg),  Bacillus  phosphoresceiis  indicus  (Fischer),  Bacillus  phosphoresccns 
indigenus  (Fischer),  feacillus  phosphoresceiis  gelidus  (Katz),  Bacillus  sina 
ragdino-phosphorescens  (Katz),  Bacillus  argenteo-phosphorescens  Nos.  I., 
II.,  and  III.  (Katz),  Bacillus  cyaneo-phosphorescens  (Katz),  Bacillus  ar- 
genteo-phosphorescens liquefaciens  (Katz),  Bacillus  ramosus,  Bacillus  sub- 
tilis  (Ehrenberg),  Proteus  sulfureus  (Lindenborn),  Bacillus  aureus  (Ada 
metz),  Bacillus  brunneus  (Adametz),  Bacillus  flavocoriaceus  (Adametx), 
Bacillus  fluorescens non-liquefaciens,  feacillus  latericeus  (Adametz),  Bacillus 
stolonatus  (Adametz),  Bacillus  berolinensis  indicus  (Classen),  Bacillus  ery- 
throsporus  (Eidam),  Bacillus  luteus  (List),  Bacillus  aquatilis  sulcatus  Nos. 
1,  2,  3,  4,  and  5  (Weichselbaum),  Bacillus  albus  (Eisenberg),  Bacillus  multi- 
ped iculosus(  Fliigge),  Bacillus  Ziirnianum  (List),  Bacillus  fulvus  (Zimmer- 
manu),  Bacillus  helvolus  (Zimmermann),  Bacillus  ochracens  (Zimmer- 


BACTERIA  IN  WATER.  649 

mann),  Bacillus  plicatus,  Bacillus  deyorans  (Zim merman n),  Bacillus  gracilis 
(Zimmerinann),  Bacillus  guttatus  (Zimmermann),  Bacillus  implexus  (Zim- 
mermann),  Bacillus  punctatus  (Zimmermann),  Bacillus  radiatus  aquatilis 
(Zimmermann),  Bacillus  vermiculosus  (Zimmermann),  Bacillus  cpnstrictus 
(Zimmermann),  Bacillus  fluorescens  aureus  (Zimmermann),  Bacillus  fluo- 
rescens  longus  (Zimmermann),  Bacillus  fluorescens  tenuis  (Zimmermann), 
Bacillus  fuscus  (Zimmermann),  Bacillus  rubefaciens  (Zimmermann),  Bacil- 
lus subflavus  (Zimmermann),  Bacillus  janthinus  (Zopf),  Bacillus  mycoides 
(Fliigge),  Bacillus  tremelloides  (Tils),  Bacillus  cuticularis  (Tils),  Bacillus 
filiformis  (Tils),  Bacillus  ubiquitus  (Jordan),  Bacillus  circulans  (Jordan), 
Bacillus  superficialis  (Jordan),  Bacillus  reticularis  (Jordan),  Bacillus  ru- 
besceiis  (Jordan),  Bacillus  hyalinus  (Jordan),  Bacillus  cloacae  (Jordan), 
Bacillus  delicatulus  (Jordan),  Bacillus  violaceus  laurentius  (Jordan). 

PATHOGENIC  BACILLI. 

Bacillus  typhi  abdominalis  (Eberth,  Gaffky),  Bacillus  erysipelatos  suis 
("  Bacillus  murisepticus, "  Koch),  Bacillus  septicsemiae  haernorrhagicae 
("Bacillus  cuniculicida,"  Koch),  Proteus  vulgaris  (Hauser),  Proteus  mira- 
bilis  (Hauser),  Bacillus  canalis  capsulatus  (Mori),  Bacillus  canalis  parvus 
(Mori),  Spirillum  cholerae  Asiaticae  ("Comma  bacillus,"  Koch),  Bacillus  coli 
communis  (Escherich),  Bacillus  hydrophilus  fuscus  (Sanarelli),  Bacillus 
venenosus  (Vaughan),  Bacillus  yenenosus  brevis  (Vaughan),  Bacillus  vene- 
nosus  invisibilis  (Vaughan),  Bacillus  venenosus  liquefaciens  (Vaughan). 

The  following  additional  species  are  described  by  Zimmermann  (1894)  in 
his  second  publication  ("Die  Bakterien  unserer  Trink-  und  Nutzwasser "). 
Micrococcus  candidus,  Micrococcus  coralloides,  Streptococcus  cinereus,  Mi- 
crococcus  sulpliureus,  Micrococcus  galbanatus,  Micrococcus  erythromyxa, 
Sarcina  flavea,  Sarcina  aurantiaca,  Sarcina  rosea.  Bacillus  ruber,  Bacillus 
miiiiaceus,  Bacillus  mesentericus  roseus,  Bacillus  carnosus,  Bacillus  chryso- 
gloia,  Bacillus  multipediculus  flavus,  Bacillus  villosus,  Bacillus  radiatus, 
Bacill us  fluorescens  al bus,  Bacillus  viridans,  Bacillus  turcosa,  Bacillus  halans, 
Bacillus  nacreaceus,  Bacillus  mirabilis,  Bacillus  umbilicatus,  Bacillus  lactis 
viscosus,  Bacillus  syiixanthus,  Bacillus  sericeus,  Bacillus  minutus,  Bacillus 
stellatus,  Bacillus  radicosus,  Bacillus  vemicosus,  Bacillus  mucosus,  Bacillus 
centralis,  Bacillus  spumosus,  Bacillus  aiinulatus,  Bacillus  liquefaciens,  Bacil- 
lus disciformans. 

The  following  spirilla  and  "  vibrios"  have  also  been  found  in  water- 
chiefly  in  river  water  : 

Spirillum  volutans,  Spirillum  sanguineum,  Spirillum  serpens,  Vibrio  ru- 
gula,  Spirillum  plicatile,  Spirillum  marinum  (Russell).  Spirillum  cholerae 
Asiaticse,  Spirillum  of  Renon,  Vibrio  aquatilis  (Gunther),  Vibrio  of  Weibel, 
Vibrios  of  Bujwid  (Bacillus  choleroides  a  and  6),  Vibrio  of  Loffler,  Vibrios 
of  Boiihoff,  Vibrio  of  Blackstein,  Vibrios  of  Sanarelli,  Vibrios  of  Fischer, 
Vibrio  Beroliiiensis,  Vibrio  Danubicus,  Vibrio  of  Pfuhl  (v.  Metchnikovi  ?). 
Several  of  the  "  vibrios"  in  this  list  which  have  recently  been  obtained  from 
river  water  in  various  parts  of  Europe  are  probably  varieties  of  the  cholera 
spirillum. 

ADDITIONAL   NOTES   UPON    BACTERIA  IN  WATER. 

It  is  now  generally  recognized  by  bacteriologists  that  the  potability  of 
water  is  to  be  determined  by  an  investigation  relating  to  the  presence  or  ab- 
sence of  known  pathogenic  bacteria,  rather  than  by  an  estimate  of  the  num- 
ber of  bacteria  present  in  each  cubic  centimetre  of  the  water  under  exami- 
nation. From  a  sanitary  point  of  view  the  most  important  of  these  pathogenic 
bacteria  are  the  cholera  spirillum  and  allied  "  vibrios,"  the  bacilli  of  the  "  ty- 
phoid group "  (Bacillus  typhi  abdominalis  and  allied  forms),  the  bacilli  of 
the  "colon  group"  (Bacillus  coli  communis  with  its  varieties  and  similar 
bacilli  of  faecal  origin).  When  one  of  these  pathogenic  bacilli  is  present  in  a 
45 


650  BACTERIA  IN  WATER. 

water-supply  in  small  numbers  as  compared  with  the  number  of  saprophytic 
bacteria,  it  is  not  an  easy  matter  to  demonstrate  the  fact  by  the  ordinary 
plate  method,  especially  in  the  case  of  non-liquefying  species  like  the  typhoid 
bacillus.  If  we  have,  for  example,  one  typhoid  bacillus  to  one  thousand  ba- 
cilli of  other  species  it  is  evident  that  in  a  series  of  three  plates,  made  in  the 
usual  way  for  the  purpose  of  obtaining  isolated  colonies,  there  would  be  but  a 
small  chance  of  obtaining  a  colony  of  the  typhoid  bacillus  in  plate  No.  3, 
and  a  plate  containing  one  thousand  colonies  or  more  would  be  so  crowded 
that  the  detection  of  the  single  typhoid  colony  would  be  very  difficult.  For 
this  reason,  it  is  necessary  to  resort  to  special  methods  by  which  the  more 
numerous  saprophytic  bacteria  will  be  excluded,  or  their  numbers  greatly 
reduced.  Some  of  the  methods  which  have  been  successfully  employed  for 
the  detection  of  the  typhoid  bacillus  and  of  the  cholera  spirillum  are  given 
in  the  sections  devoted  to  these  microorganisms.  We  give  below  some  de- 
tails relating  to  the  methods  employed  by  bacteriologists  of  recognized  com- 
petence in  recent  investigations  : 

Marpmann  (1895)  considers  all  water  which  contains  faecal  bacteria  as 
dangerous  as  a  supply  for  drinking  purposes.  For  the  detection  of  patho- 
genic bacteria  he  recommends  the  following  procedure : 

The  pathogenic  bacteria  are  divided  into  two  groups  by  cultivation  in  nu- 
trient agar  containing  0.2  percent  of  citric  acid,  and  in  the  same  medium  con- 
taining two  per  cent  of  sodium  carbonate.  The  bacilli  of  the  typhoid  group 
are  said  to  grow  in  the  acid  medium  but  not  in  that  containing  two  per  cent 
of  sodium  carbonate.  On  the  other  hand,  cholera  vibrios  develop  in  the  al- 
kaline medium  but  not  in  that  containing  0.2  per  cent  of  citric  acid.  The  ba- 
cilli of  the  colon  group  also  ("  cloaca-bacilli")  do  not  grow  in  the  medium 
containing  citric  acid.  Bouillon  containing  the  same  amounts  of  acid  and 
alkali  is  also  employed.  The  water  to  be  examined  is  first  mixed  with  an 
equal  portion  of  acid  and  of  alkaline  bouillon  in  two  test  tubes,  and  these  are 
kept  at  a  temperature  of  30°  C.  for  twenty -four  hours,  during  which  time 
the  pathogenic  bacteria,  if  present,  will  multiply  and  cause  a  clouding  of  the 
culture  media.  Inoculations  are  now  made  into  the  acid  and  alkaline  agar 
and  gelatin.  Growth  in  alkaline  gelatin  at  the  room  temperature  (10°  to  18° 
C.)  is  due  to  "cloaca-bacteria"  ;  growth  in  acid  gelatin  at  20°  to  23°  C.  is  due 
to  bacilli  of  the  typhoid  group.  Plates  should  also  be  made  from  the  clouded 
bouillon,  acid  and  alkaline  ;  and  the  colonies  resembling  those  of  the  typhoid 
or  of  the  colon  group  should  be  tested  in  nutrient  gelatin  containing  sugar 
to  ascertain  whether  there  is  development  of  gas,  in  which  case  the  bacilli  are 
of  the  colon  group. 

When  typhoid  and  colon  bacilli  are  associated  in  water  the  last-mentioned 
bacillus  takes  the  precedence,  and  the  typhoid  bacillus  has  a  tendency  to  dis- 
appear. This  is  shown  by  the  experiments  of  Girnbert  (1894),  who  introduced, 
at  the  same  time,  colon  bacilli  and  typhoid  bacilli  into  water,  and  found  that 
at  the  end  of  forty-eight  hours  he  was  no  longer  able  to  isolate  the  typhoid 
bacillus  from  plates.  In  view  of  this  fact  failure  to  find  the  typhoid  bacillus 
does  not  relieve  the  water  from  the  suspicion  of  being  dangerous  if  the  colon 
bacillus  is  present.  But,  on  the  other  nand,  this  bacillus  is  so  common  that 
it  is  perhaps  the  exception  when  it  is  not  present  in  surface  waters.  As 
pointed  out  by  von  Freudenreich  (1895)  it  may,  however,  escape  detection 
unless  a  considerable  quantity  of  water  is  used  in  making  the  test.  When 
the  quantity  is  from  one  hundred  to  five  hundred  cubic  centimetres,  in- 
stead of  from  one  to  five  cubic  centimetres,  as  was  formerly  the  usual  amount 
employed,  it  is  found  not  infrequently  even  in  spring  water  (von  Freuden- 
reich). 

The  author  last  mentioned  says  that  when  present  in  small  numbers  it 
may  be  demonstrated  by  the  method  of  Vincent,  as  follows  :  Mix  of  the  water 
ninety  cubic  centimetres  with  ten  cubic  centimetres  of  a  twenty-per-cent 
solution  of  peptone,  and  one  cubic  centimetre  of  a  seven-per-cent  solution  of 
carbolic  acid ;  place  in  the  incubating  oven  at  42°  C.  If  development  oc- 


BACTERIA  IN  WATER.  651 

curs  it  will  probably  be  due  to  the  colon  bacillus,  but  it  will  be  necessary  to 
make  plates  and  pure  cultures  from  single  colonies  in  order  to  determine 
this  with  certainty.  The  demonstration  may  be  made  more  quickly,  accord- 
ing to  von  Freudenreich,  by  using  a  medium  containing  milk  sugar  (five  per 
cent)  and  cultivating  at  35°  C.  If  the  colon  bacillus  is  present  there  will  be 
an  abundant  development  of  gas  in  from  twelve  to  twenty -four  hours,  and 
the  bacillus  may  then  be  readily  isolated  by  the  plate  method.  The  colon 
bacillus  has  been  found  byMoissan  and  Gimbert  in  mineral  waters  bottled  in 
France.  Poncet  (1895)  has  made  a  careful  study  of  the  bacteria  found  in  the 
various  springs  at  Vichy.  The  species  described  are  all  harmless  water  bac- 
teria and  have  little  interest  from  a  sanitary  point  of  view. 

Kruse  (1894),  as  a  result  of  his  extended  researches  and  of  a  critical  con- 
sideration of  the  experimental  data  available,  arrives  at  the  conclusion  that  a 
sanitary  inspection  of  the  sources  of  supply  is  more  important,  in  determin- 
ing the  safety  of  the  supply  from  a  sanitary  point  of  view,  than  a  chemical 
or  bacteriological  examination.  The  writer  has  for  some  years  past  enter- 
tained the  same  opinion.  Kruse  says,  however,  that  for  the  control  of  fil- 
tering plants  bacteriological  "counting-methods"  are  indispensable.  He 
also  ascribes  a  "high  scientific  value"  to  investigations  relating  to  the  pres- 
ence of  the  more  important  pathogenic  bacteria;  but  says  that,  notwith- 
standing the  improvements  in  methods  of  research,  we  cannot  wait  for  a 
demonstration  of  the  presence  of  the  cholera  or  typhoid  bacteria  before  con- 
demning a  water  as  probably  unsafe,  if  sources  of  contamination  are  dis- 
covered— or,  we  would  add,  if  cases  of  cholera  or  typhoid  fever  can  be  traced 
with  a  fair  degree  of  certainty  to  the  use  of  water  from  a  given  source. 

Fischer  (1894),  in  his  account  of  the  researches  made  during  the  Plankton 
expedition,  has  given  a  summary  of  the  experimental  evidence  relating  to  the 
presence  of  bacteria  in  the  waters  of,  the  ocean.  The  species  found  were  for 
the  most  part  different  from  those  found  in  lakes  and  rivers,  and  at  some 
distance  from  the  shore  none  of  the  previously  known  species  of  micrococci 
and  bacilli  were  encountered.  The  number  of  bacteria  in  samples  from  the 
surface  at  a  distance  from  the  shore  was  comparatively  small  (usually  less 
than  five  hundred  per  cubic  centimetre),  but  in  the  vicinity  of  land  very 
large  numbers  were  sometimes  found.  At  a  distance  of  ten  metres  below  the 
surface  the  number  found  was  greatly  in  excess  of  the  number  at  the  surface 
—the  difference  being  probably  due  to  the  germicidal  action  of  sunlight.  At 
depths  of  four  hundred  metres  bacteria  were  constantly  found  in  great  num- 
bers, and  water  from  a  depth  of  eleven  hundred  metres  was  still  found  to 
contain  them. 


III. 

BACTERIA    IN  THE  SOIL. 

SURFACE  soil,  and  especially  that  which  is  rich  in  organic  matter, 
contains  very  numerous  bacteria  of  many  different  species.  Some  of 
these  are  of  special  interest  on  account  of  their  pathogenic  power. 
Thus  the  bacillus  of  malignant  oedema  and  the  bacillus  of  tetanus 
have  been  shown  to  be  widely  distributed  species,  which  have  been 
obtained  by  investigators  in  various  parts  of  the  world  by  inoculating 
susceptible  animals — guinea-pigs  or  mice — with  a  little  rich  surface 
soil.  Other  species  are  interesting  because  of  their  action  in  nitrifi- 
cation and  in  the  destructive  decomposition  of  organic  material  by 
which  it  is  fitted  for  assimilation  by  the  higher  plants.  Many  of  the 
bacteria  present  in  the  soil  are  strictly  anaerobic,  and  in  attempts  to 
estimate  the  number  and  kind  of  microorganisms  present  in  a  given 
sample  this  fact  must  be  kept  in  view. 

The  simplest  method  of  studying  the  bacteria  in  the  soil  consists 
in  introducing  a  small  quantity  into  liquefied  gelatin  in  test  tubes, 
and,  after  carefully  crushing  it  with  a  sterilized  glass  rod  and  thor- 
oughly mixing  it  with  the  gelatin,  making  roll  tubes  in  the  usual 
way.  Some  of  these  should  be  put  up  for  anaerobic  cultures — i.e., 
the  tube  should  be  filled  with  an  atmosphere  of  hydrogen  according 
to  FrankeFs  method.  If  the  object  in  view  is  to  estimate  the  num- 
ber of  bacteria  in,  a  given  sample  of  soil  the  difficulty  is  encountered 
that,  however  finely  crushed,  the  little  masses  of  earth  are  likely  to 
contain  numerous  bacteria,  and  we  cannot  safely  assume  that  each 
colony  originates  from  a  single  germ.  Thoroughly  washing  a  small 
quantity  of  soil,  by  agitation,  in  a  considerable  quantity  of  distilled 
water,  and  then  adding  a  definite  quantity  of  the  water  to  nutrient 
gelatin  and  making  roll  tubes  or  plates,  as  in  water  analysis,  sug- 
gests itself  as  a  simple  method  ;  but  Friinkel  has  shown  that  it  is  far 
from  being  reliable  when  the  object  is  to  estimate  the  number  of 
bacteria.  He  obtained  more  uniform  and  accurate  results  by  intro- 
ducing the  earth  at  once  into  liquefied  gelatin  arid  crushing  it  as 
thoroughly  as  possible  with  a  strong  platinum  wire,  after  which  as 
thorough  a  mixture  as  possible  was  effected  by  tilting  the  tube  up 


BACTERIA   IN   THE   SOIL.  653 

and  down.  But  for  the  purpose  of  obtaining  pure  cultures  from  sin- 
gle colonies  of  the  various,  species  present,  we  should  prefer  to  wash 
the  earth  in  distilled  water  and  to  allow  the  sediment  to  settle  before 
taking  a  portion  of  the  water  to  add  to  the  nutrient  medium. 

In  some  experiments  made  in  1881  Koch  ascertained  that  in  soil 
which  had  not  been  disturbed  but  few  bacteria  were  to  be  found  at 
the  depth  of  a  metre;  and  this  fact  has  since  been  established  by  Ihe 
extended  researches  of  Frankel,  who  devised  a  special  boring  instru- 
ment for  obtaining  samples  of  earth  from  different  depths.  Miquel, 
in  1879,  estimated  the  number  of  bacteria  in  one  gramme  of  earth 
collected  in  the  park  of  Montsouri,  Paris,  at  a  depth  of  twenty  centi- 
metres, at  700,000;  and  in  a  cultivated  field  which  had  been  treated 
with  manure,  at  900,000.  The  following  results  were  obtained  by 
Adametz  :  One  gramme  of  earth  from  a  sandy  soil  contained  at  the 
surface  380,000,  at  a  depth  of  twenty  to  twenty-five  centimetres 
400,000 ;  the  same  quantity  of  clayey  soil  contained  at  the  surface 
500,000,  at  a  depth  of  twenty  to  twenty-five  centimetres  460,000. 

In  experiments  made  by  Beumer  (1886)  and  by  Maggiora  (1887) 
considerably  greater  numbers  were  found,  but  the  last-named  ob- 
server, in  some  instances  at  least,  kept  the  earth  for  some  time  after 
collecting  it,  which  may  have  materially  influenced  the  result. 
Beumer  obtained  from  a  specimen  of  sandy  humus  taken  from  a 
depth  of  three  metres  45,000,000  to  the  gramme ;  at  four  metres, 
10,000,000;  at  five  metres,  8,000,000;  at  six  metres,  5,000,000. 
These  specimens  were  obtained  from  the  vicinity  of  hospitals  at 
Greifswald.  In  a  churchyard,  at  a  depth  of  four  metres,  the  num- 
ber in  one  experiment  was  1,152,000,  and  in  another  1,278,000. 

Frankel  has  given  special  attention  to  the  examination  of  undis- 
turbed soil  not  in  the  immediate  vicinity  of  dwellings.  In  samples 
from  a  fruit  orchard  near  Potsdam  he  found  that  the  superficial 
layers  contained  from  50,000  to  350,000  germs  per  cubic  centimetre. 
The  greatest  number  was  not  immediately  upon  the  surface,  but  at 
from  one-quarter  to  one-half  metre  below  the  surface.  The  num- 
ber was  found  to  be  greater  in  summer  than  in  winter,  the  maximum 
being  in  July  and  August.  At  a  depth  of  three-quarters  of  a  metre 
to  a  metre  and  a  half  there  was  a  very  great  and  abrupt  diminution  in 
the  number  of  germs.  From  200, 000  at  one-half  metre  the  number  fell 
to  2,000  at  a  depth  of  a  metre,  from  250,000  at  three-quarters  of  a 
metre  to  200  at  one  metre,  etc. ,  and  at  a  depth  of  one  and  one-half 
metres,  in  some  instances,  no  more  living  germs  were  obtained.  In 
other  experiments  a  few  colonies  developed  from  earth  obtained  at  a 
depth  of  three  or  four  metres,  but  these  were  slow  in  making  their 
appearance,  and  often  several  days,  or  even  weeks,  elapsed  before 
they  became  visible  in  Esmarch  roll  tubes.  In  experiments  with  sur- 


654  BACTERIA   IN   THE   SOIL. 

face  soil,  on  the  contrary,  a  multitude  of  colonies  developed  within 
twenty-four  to  forty -eight  hours,  and,  as  many  liquefying  bacteria 
were  present,  it  was  necessary  to  make  the  enumeration  on  the  first 
or  second  day,  at  which  time,  no  doubt,  many  of  the  bacteria  present 
had  not  yet  formed  visible  colonies.  The  results  obtained  have, 
therefore,  only  a  relative  value. 

The  most  important  fact  developed  by  FrankeFs  researches  is  that 
in  virgin  soil  there  is  a  dividing  line  at  a  depth  of  from  three-quarters 
to  one  and  one-half  metres,  below  which  very  few  bacteria  are  found, 
and  that,  consequently,  the  "  ground- water  region  "  is  free  from  micro- 
organisms, or  nearly  so,  notwithstanding  the  immense  numbers  pre- 
sent in  the  superficial  layers. 

The  extended  researches  of  Maggiora,  made  in  the  vicinity  of 
Turin,  led  him  to  the  following  conclusions  : 

1.  The  number  of  germs  in  desert  and  forest  soils  is  much  smaller,  other 
conditions  being  equal,  than  in  cultivated  lands,  and  in  these  it  is  less  than 
in  inhabited  localities. 

2.  In  desert  soils  the  number  of  germs  bears  a  relation  (a)  to  the  geologi- 
cal epoch  to  which  the  lands  belong,  and,  within  certain  limits,  to  the  heigtit 
above  the  level  of  the  sea — the  older  the  soil  and  the  greater  the  altitude, 
other  things  being  equal,  the  fewer  the  germs  ;  (&)  to  the  compactness  and 
aeration  of  the  soil — the  more  compact  and  impermeable  to  air  the  smaller 
the  number  of  germs  capable  of  developing  in  gelatin  ;  (c)  to  the  nature  of 
the  soil — sandy  soils  contain  fewer  germs  than  soils  rich  in  clay  and  in 
humus. 

3.  In  cultivated  lands  the  number  of  germs  augments  with  the  activity 
of  cultivation  and  the  strength  of  the  fertilizers  used. 

4.  In  inhabited  localities  the  number  of  germs  in  the  superficial  layers  is 
very  great.     In  the  deep  layers  it  usually  diminishes  rapidly,  as  is  the  case 
in  all  other  soils. 

As  to  the  kinds  of  bacteria  present,  and  their  biological  characters 
and  functions  in  preparing  organic  material  for  assimilation  by  the 
plants  whose  roots  penetrate  the  soil,  we  have  yet  much  to  learn. 
Frankel  remarks  that  the  species  most  frequently  encountered  in  the 
deeper  strata  of  the  soil  were  three  bacilli  which  also  abound  in  the 
superficial  layers — viz.,  the  "  hay  bacillus,"  the  "wurzel  bacillus," 
and  the  "hirnbacillus."  In  all  eleven  bacilli  were  isolated  and  cul- 
tivated. Micrococci  were  only  found  four  times,  and  spirilla  not  at 
all.  Mould  fungi  were  more  abundant,  and  especially  one  previously 
obtained  from  the  air  by  Hesse  and  called  by  him  "brauner  Schim- 
melpilz."  Anaerobic  bacilli,  contrary  to  expectation,  were  not  ob- 
tained in  FrankePs  researches,  and  no  pathogenic  species  were  found 
in  the  deeper  layers  of  the  soil.  We  have  already  referred  to  the 
fact  that  the  bacillus  of  malignant  oedema  and  the  bacillus  of  tetanus, 
two  pathogenic,  anaerobic  species,  are  common  in  rich  surface  soil  in 
various  parts  of  the  world. 


BACTERIA   IN   THE   SOIL.  655 

The  results  obtained  in  the  researches  referred  to,  in  which  nutri- 
ent gelatin  was  used  as  a  culture  medium,  are  no  doubt  very  in- 
complete, not  only  on  account  of  the  liquefaction  of  the  gelatin  by 
common  liquefying  bacilli  before  other  species  present  have  formed 
visible  colonies,  but  also  because  this  is  not  a  favorable  culture  me- 
dium for  some  of  the  species  present  in  the  soil.  Thus  Frankland  has 
succeeded  in  isolating  a  nitrifying  ferment  which  he  calls  "  Bacillo- 
coccus,"  which  grows  abundantly  in  bouillon,  but  fails  to  grow  in 
nutrient  gelatin.  Winogradski  has  also  obtained  in  pure  cultures  a 
nitrifying  ferment  from  the  soil  in  the  vicinity  of  Zurich,  which  he 
has  called  "  Nitromonas." 

Comparatively  few  micrococci  are  found  in  the  soil,  while  in  the 
air  they  are  usually  found  to  be  more  abundant  than  bacilli.  This 
is  perhaps  due  to  the  fact  that  the  bacilli  are  more  promptly  destroyed 
by  desiccation  and  the  action  of  sunlight. 

Several  bacteriologists  have  made  investigations  relating  to  the 
duration  of  vitality  of  pathogenic  bacteria  in  the  soil.  Frankel  found 
that  in  Berlin  the  bacillus  of  anthrax,  in  Esmarch  roll  tubes,  when 
buried  in  the  soil  at  a  depth  of  two  metres,  only  occasionally  gave 
evidence  of  growth,  and  at  three  metres  no  development  occurred. 
The  comparatively  low  temperature  at  this  depth  was  no  doubt  an 
important  factor  in  influencing  the  result.  The  cholera  spirillum  in 
the  months  of  August,  September,  and  October  grew  at  a  depth  of 
three  metres,  but  in  the  remaining  months  of  the  year  failed  to  grow 
at  two,  while  growth  occurred  at  one  and  one-half  metres.  The 
bacillus  of  typhoid  fever  grew  at  three  metres  during  the  greater 
portion  of  the  year. 

Giaxa  has  made  extended  and  interesting  experiments  with  the 
cholera  spirillum,  cultures  of  which  he  added  to  different  kinds  of 
soil  (garden  earth,  clay,  sand)  and  placed  at  different  depths  below 
the  surface — one-quarter,  one-half,  and  one  metre.  Some  of  the  earth 
was  sterilized  and  some  was  not.  In  the  unsterilized  earth  he  found 
the  cholera  spirillum  in  considerable  numbers  at  the  end  of  twenty- 
four  hours  at  the  greatest  depth  tested  (one  metre),  but  at  the  end  of 
forty- eight  hours  it  had  disappeared  in  five  experiments  out  of  seven 
— the  lowest  temperature  at  this  depth  was  20°  C.  In  the  sterilized 
soil  the  result  was  different ;  the  cholera  spirillum  was  present  in 
enormous  numbers  at  the  end  of  four  days  at  a  depth  of  a  metre, 
and  was  still  found  in  smaller  numbers  at  the  end  of  twelve  days,  but 
had  disappeared  at  the  end  of  twenty-one  days.  These  results  indicate 
that  the  presence  of  common  saprophytes  in  the  soil  is  prejudicial  to 
the  development  of  the  cholera  spirillum,  and  that  under  ordinary 
circumstances  it  succumbs  in  the  struggle  for  existence  with  these 
more  hardy  microorganisms. 


656  BACTERIA   IN    THE  SOIL. 

The  researches  of  Proskauer  (1891)  confirm  those  of  Frankel  and 
others  as  to  the  rapid  diminution  in  the  number  of  bacteria  in  the 
deeper  layers  of  the  soil.  They  also  agree  with  those  of  Gartner  in 
showing  that  in  the  soil  of  churchyards  the  number  of  bacteria 
diminishes  greatly  in  the  soil  beneath  the  layer  containing  coffins. 
In  general  the  influence  of  dead  bodies  upon  the  bacteria  in  the  soil 
in  the  vicinity  of  coffins  was  very  slight ;  in  the  subsoil  of  the  grave- 
yard there  were  not  many  more  bacteria  than  in  similar  soil  outside 
of  this.  Reimers  had  previously  shown  that  samples  of  earth  from 
two  graves,  in  one  of  which  the  body  had  been  buried  for  thirty-five 
years  and  in  the  other  for  one  and  one-half  years,  gave  similar  re- 
sults when  examined  by  bacteriological  methods. 

Manfredi  in  1892  published  the  results  of  his  extended  investiga- 
tions relating  to  the  dust  in  the  streets  of  Naples.  The  number  of 
bacteria  varied  greatly  in  different  parts  of  the  city.  In  streets 
where  the  traffic  was  least  and  hygienic  conditions  the  best  the 
average  number  was  10,000,000  per  gramme.  In  dirty  and  busy 
thoroughfares  the  average  was  1,000,000,000,  and  in  certain  locali- 
ties the  number  was  even  five  times  as  great  as  this.  Injections  into 
guinea-pigs  gave  a  positive  result  in  seventy-three  per  cent  of  the 
animals  experimented  upon.  Among  the  known  pathogenic  bacte- 
ria obtained  in  this  way  were  the  pus  cocci  (in  eight),  Bacillus  tuber- 
culosis (in  three),  the  bacillus  of  malignant  oedema,  and  the  tetanus 
bacillus. 

In  the  memoir  of  Fiilles  (1891)  the  following  species  are  described 
as  having  been  found  by  him  in  the  soil  at  Freiburg,  Germany: 


MICROCOCCI. 

(a)  Non-liquefying. — Micrococcus  aurantiacus  (Cohn),  Micrococcus  can- 
us  (Cohn),  Micrococcus  luteus  (Cohn),  Micrococcus  candicans  (Flugge), 
Micrococcus  versicolor  (Fliigge),  Micrococcus  cinnabareus  (Flugge),  Micro- 


didus  (Cohn),  Micrococcus  luteus  (Colin),  Micrococcus  candicans  (Fliigge) 

ugge),  Micro 
coccus  cereus  albus  (Passet),  Micrococcus  fervitosus  (Adametz),  Bother  coc 


cus  (Maschek). 

(b)  Liquefying.  —  Micrococcus  flavus  liquefaciens  (Flugge),  Micrococcus 
flavus  desidens  (Flugge),  Diplococcus  luteus  (Adametz),  Sarcina  lutea. 

NON-PATHOGENIC  BACILLI. 

(a)  Non-liquefying.  —  Bacillus  fluorescens  putidus  (Flugge),  Bacillus  mus- 
coides  (Liborius),  Bacillus  scissus  (Frankland),  Bacillus  candicans,  Bacillus 
diffusus   (Frankland),  Bacillus  fihformis   (Tils),  Bacillus  luteus  (Flugge), 
Fluorescent  water  bacillus  (Eisenberg),  Bacillus  viridis  pallescens  (Frick), 
Bluish-green  fluorescent  bacillus  (Adametz),  Bacillus  stolonatus  (Adametz), 
Bacillus  Ziirnianum  (List),  Bacillus  aerogenes  (Miller),  Bacillus  No.  1  and 
Bacillus  No.  2  (Fiilles). 

(b)  Liquefying.—  Bacillus  ramosus  liquefaciens  (FliiggeX  Bacillus  liqui- 
dus    (Frankland),    Bacillus  ramosus  —  "wurzel  bacillus,     Bacillus  subtilis 


BACTERIA   IN  THE   SOIL.  657 

(Ehrenberg),  Bacillus  mesentericus  fus.cus  (Fliigge),  Bacillus  mesentericus 
vulgatus  (Fliigge),  Bacillus  fluorescens  liquefaciens  (Fliigge),  Lemon-yellow 
bacillus  (Maschek),  Green  yellow  bacillus  (Eisenberg),  Gas-forming  bacillus 
(Eisenberg),  Gray  bacillus  (Maschek),  Bacillus  prodigiosus  (Ehrenberg), 
Proteus  mirabilis  (Hauser),  Proteus  vulgaris  (Hauser),  Bacillus  mesentericus 
vulgatus,  Bicillus  cuticularis  (Tils),  "  Weisser  bacillus"  (Eisenberg). 

(c)  Pathogenic. — Bacillus  oadematis  maligni  (Koch). 

In  addition  to  the  above  the  following  species  have  been  described  by 
other  authors:  Bacillus  liquefaciens  magnus  (Liideritz),  Bacillus  radiatus 
(Liideritz),  Bacillus  solidus  (Liideritz),  Bacillus  mycoides  roseus  (Scholl), 
Bacillus  viscosus  (Frankland),  Bacillus  candicans  (Frankland),  Bacillus 
poliformis  (Liborius),  Clostridium  foatidum  (Liborius). 

Pathogenic  species. — Staphylococcus  pyogenes  aureus  (Rosenbach),  Ba- 
cillus tetani  (Nicolaier),  Streptococcus  septicus  (Nicolaier),  Pseudo-oedema  ba- 
cillus (Liborius),  Bacillus  septicus  agrigenus  (Nicolaier),  Bacillus  of  Utpadel. 


IV. 

BACTERIA  OF  THE  SURFACE  OF  THE  BODY  AND  OF 
EXPOSED  MUCOUS  MEMBRANES. 

GREAT  numbers  of  bacteria  of  various  species  multiply  upon  the 
surface  of  the  human  body,  where  they  find  the  necessary  pabulum 
in  the  excretions  from  the  skin  and  the  exfoliated  epithelium.  Evi- 
dently the  number  will  be  largely  influenced  by  the  clothing  worn, 
the  atmospheric  conditions  as  to  heat  and  moisture,  personal  habits, 
etc.  The  writer  has  frequently  inoculated  culture  media  with  a  drop 
of  sterilized  fluid  which  had  been  placed  upon  the  surface  of  the  body 
of  patients  in  hospitals  and  of  healthy  persons.  By  friction  with  a 
platinum  needle  at  the  point  where  the  drop  of  fluid  is  applied  the 
surface  is  washed  and  a  little  epithelium  detached.  Cultures  may 
always  be  obtained  by  inoculating  nutrient  media  from  a  drop  of  fluid 
applied  in  this  way.  Micrococci  of  various  species,  including  the  pus 
cocci,  are  very  commonly  encountered  ;  sarcinse  and  various  bacilli 
are  also  frequently  met  with.  Even  the  hands,  which  by  reason  of 
their  exposure  and  frequent  ablutions  are  freer  from  exfoliated  epi- 
thelium than  portions  of  the  body  covered  with  clothing,  have  con- 
stantly attached  to  their  surface  a  considerable  number  of  bacteria. 
This  is  shown  by  the  experiments  of  Kummel  and  Forster,  of  Fiir- 
bringer  and  others,  with  reference  to  the  disinfection  of  the  hands, 
Forster  found  that  after  the  most  careful  cleaning  of  the  hands  with 
soap,  water,  and  a  brush,  contact  of  the  fingers  with  nutrient  gelatin 
always  resulted  in  the  development  of  a  greater  or  less  number  of 
colonies. 

Bordoiii-Uffreduzzi,  in  his  researches  relating  to  the  bacteria  of 
the  skin,  obtained  in  pure  cultures  five  different  species  of  micrococci 
and  two  bacilli.  Pure  cultures  of  his  Bacterium  graveolens,  which 
was  usually  found  between  the  toes,  gave  off  a  disagreeable  odor  like 
that  observed  from  this  locality  in  certain  individuals.  In  his  re- 
searches made  in  Havana  the  writer  frequently  encountered  in  cul- 
tures from  the  surface,  associated  with  various  micrococci,  his  Micro- 
coccus  tetragenus  versatilis. 

Fiirbringer  found  quite  frequently  in  the  spaces  beneath  the  fin- 


BACTERIA  OF  THE  SURFACE  OF  THE  BODY        659 

ger  nails  Staphylococcus  pyogenes  aureus  associated  with  various 
other  microorganisms.  A  similar  result  had  previously  been  reported 
by  Bockhart. 

In  his  examinations  of  water  from  various  sources  Miquel  found 
that  "wash- water"  from  the  floating  laundries  on  the  Seine  con- 
tained more  bacteria  than  water  from  any  other  source,  even  than 
the  water  of  the  Paris  sewers.  His  enumeration  gave  twenty-six 
million  germs  per  cubic  centimetre. 

Hohein  has  enumerated  the  colonies  developing  from  undercloth- 
ing worn  for  various  lengths  of  time  and  made  of  different  kinds  of 
material.  A  piece  of  the  goods  to  be  tested  was  sewed  fast  to  the 
underclothing,  so  as  to  come  in  immediate  contact  with  the  body  ;  at 
the  end  of  a  given  time  a  fragment  one-quarter  of  a  centimetre  square 
was  cut  up  as  fine  as  possible  and  distributed  in  nutrient  gelatin. 
Plates  were  made  and  the  colonies  counted  at  the  end  of  five  or  six 
days. 

In  an  experiment  in  which  sterilized  woven  goods  were  worn  next 
to  the  skin  of  the  upper  arm  the  following  results  were  obtained  : 
Linen  goods,  at  the  end  of  one  day  28,  two  days  4,180  colonies  ;  cot- 
ton goods,  end  of  one  day  105,  end  of  two  days  1,870  ;  woollen  goods, 
end  of  one  day  606,  end  of  two  days  6,799.  When  the  material  had 
been  in  contact  with  the  skin  for  four  days  the  colonies  which  devel- 
oped were  so  numerous  that  they  could  not  be  counted. 

Maggiora  isolated  twenty-two  species  of  bacteria  from  his  cultures 
inoculated  with  epidermis  from  the  foot.  None  of  these  proved  to 
be  pathogenic  for  mice,  rabbits,  or  guinea-pigs.  Several  gave  off  a 
strong  odor  of  trimethylamin,  similar  to  that  of  sweating  feet. 

The  following  species  have  been  found  upon  the  surface  of  the 
body : 

Non-pathogenic. — Diplococcus  albicans  tardus  (Unna  and  Toramasoli), 
Diplococcus  citreus  liquefaciens  (Unna  and  Tommasoli),  Diplococcus  flavus 
liquefaciens  tardus  (Unna  and  Tommasoli),  Staphylococcus  viridis  flaves- 
cens  (G-uttmann),  Bacillus  graveolens  (Bordoni-Uffreduzzi),  Bacillus  epider- 
midis  (Bordoni),  Ascobacillus  citreus  (Unna  and  Tommasoli),  Bacillus  fluo- 
rescens  liquefaciens  minutissimus  (Unna  and  Tommasoli),  Bacillus  aureus 
(Unna  and  Tommasoli),  Bacillus  ovatus  minutissimus  (Unna  and  Tomma- 
soli), Bacillus  albicans  pateriformis  (Unna  and  Tommasoli),  Bacillus  spini- 
ferus  (Unna  and  Tommasoli),  Bacillus  of  Scheurlen,  Micrococcus  tetrageiius 
versatilis  (Sternberg),  Bacillus  Havaniensis  liquefaciens  (Sternberg) . 

Pathogenic.— Staphylococcus  pyogenes  albus,  Staphylococcus  pyogenes 
aureus,  Streptococcus  pyogenes,  Diplococcus  of  Demme,  Bacillus  of  temme, 
Bacillus  of  Schimmelbusch,  Bacillus  of  Tommasoli,  Bacillus  saprogeues  II. 
(Roseiibach),  Bacillus  parvus  ovatus  (Loffler). 

SURFACE   OF   MUCOUS   MEMBRANES. 

Cultures  made  from  the  conjunctive  of  healthy  persons  usually 
show  the  presence  of  various  micrococci,  and  sometimes  of  bacilli. 


600         BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

McFarland  (1895)  says  that  in  his  researches  the  microorganisms 
found  were  for  the  most  part  "  those  already  described  by  others  and 
of  common  occurrence  in  the  air."  He  encountered,  however,  sev- 
eral bacilli  not  previously  described  ("Bacillus  hirsutus,  Bacillus 
coerulefaciens,  Bacillus  circumscriptus,  Bacillus  succinacius,  Bacillus 
violaceus  flavus").  Lachowicz  (1895)  failed  to  obtain  any  bacteria 
in  his  cultures  from  theconjunctival  sac  in  sixty-nine  per  cent  of  the 
healthy  eyes  examined  by  him  (sixty-three  eyes  in  all).  He  con- 
cludes that  the  microorganisms,  which  at  times  are  found  in  the 
healthy  con junctival  sac,  come  principally  from  the  air;  that  they 
are  present  in  small  numbers  and  probably  remain  only  for  a  short 
time.  His  experiments  show  that  most  species  when  artificially 
introduced  rapidly  diminish  in  numbers  and  soon  disappear  entirely. 
Cultures  of  Streptococcus  pyogenes  and  of  Bacillus  xerosis  conjunc- 
tive introduced  into  healthy  eyes  did  not  cause  the  slightest  irrita- 
tion. In  this  connection  we  may  remark  that  the  same  is  true  as 
regards  pathogenic  bacteria  introduced  into  the  bladder,  but  that 
when  there  is  some  cause  of  local  irritation  or  injury  a  chronic 
cystitis  is  likely  to  be  developed.  In  like  manner,  we  believe,  chronic 
conjunctivitis  may  be  developed  as  the  result  of  local  irritation  in 
connection  with  the  presence  of  pathogenic  bacteria  and  especially  of 
the  pyogenic  micrococci. 

The  extended  researches  of  Bach  (1894)  gave  results  corresponding 
with  those  of  previous  investigators,  and  not  with  those  reported  by 
Lachowicz,  who,  as  stated  above,  failed  to  obtain  cultures  from  sixty- 
nine  per  cent  of  the  healthy  eyes  examined.  Bach  says:  "  In  a  large 
percentage  of  the  cases  the  presence  of  bacteria  may  be  demonstrated, 
even  when  the  conjunctiva  presents  a  perfectly  normal  appearance ; 
the  conjunctival  sac  must  therefore  be  regarded  as  constantly  in- 
fected." Bach  describes  twenty-seven  different  microorganisms  ob- 
tained by  him  in  pure  cultures  from  this  source,  of  these  eighteen 
are  micrococci.  He  recognizes  the  fact  that  most  of  them  come  from 
the  air,  while  others  are  introduced  by  the  hands  in  rubbing  the 
eyes,  etc.  In  diseased  conditions  these  are  more  numerous  than  in 
health,  but  the  pus  cocci  are  not  infrequently  found  in  healthy  eyes. 

As  bacteria  are  constantly  present  in  the  air,  they  are  necvssarilv 
deposited  upon  the  moist  mucous  membrane  of  the  nose  during  in- 
spiration. Indeed,  it  would  appear  as  if  an  important  function  of 
this  extended  mucous  membrane  is  to  purify  the  air  from  suspended 
particles,  and  it  has  been  shown  by  experiment  that  expired  air  is 
practically  free  from  bacteria.  The  greater  number  of  those  con- 
tained in  inspired  air  are  deposited  upon  the  mucous  membrane  of 
the  anterior  nares.  In  culture  experiments  made  by  Von  Besser, 
Wright,  and  others  the  nasal  mucus  was  found  to  contain  a  great 


AND   OF   EXPOSED   MUCOUS   MEMBRANES.  G61 

variety  of  bacteria;  among  others  the  pus  cocci  were  frequently 
found  by  both  of  the  observers  mentioned.  In  eighty  one  cases  Von 
Besser  found  the  "  diplococcus  pneumoniaa  "  fourteen  times,  Staphy- 
lococcus  pyogenes  aureus  fourteen  times,  Streptococcus  pyogenes 
seven  times,  and  Friedlander's  bacillus  twice.  Twenty-eight  of  the 
cases  examined  were  convalescents  in  hospital;  among  these  the 
pathogenic  species  mentioned  were  found  less  frequently  than  in  other 
individuals.  The  following  non-pathogenic  species  were  isolated: 
Micrococcus  liquefaciens  albus  in  twenty-two  cases,  Micrococcus  al- 
bus  in  nine  cases,  Micrococcus  cumulatus  tennis  in  fourteen  cases, 
Microcqccus  flavus  liquefaciens  in  three  cases,  Bacillus  striatus  albus 
in  ten  cases,  etc. 

Paulsen  (1890)  made  thirty-one  cultures  in  nutrient  gelatin  from 
sixteen  persons  and  thirty-three  in  nutrient  agar  from  twenty-two 
persons,  with  the  following  result :  Eleven  remained  sterile,  nineteen 
showed  not  more  than  ten  colonies,  sixteen  less  than  one  hundred, 
twelve  more  than  one  hundred,  and  in  six  the  number  was  so  great 
that  they  could  not  be  counted.  Micrococci  were  more  numerous 
than  bacilli ;  of  these  a  "  sulphur-yellow  coccus  "  in  tetrads  was  found 
in  eight  individuals.  Various  species  of  liquefying  cocci,  resem- 
bling the  pus  cocci,  were  isolated,  but  the  conclusion  was  reached 
that  none  of  these  were  identical  with  the  staphylococci  of  pus, 
which  Von  Besser  and  Wright  both  found  in  a  considerable  propor- 
tion of  the  culture  experiments  made  by  them. 

Thomson  and  Hewlett  (1895)  have  recently  reported  results  which 
differ  to  some  extent  from  those  previously  reported.  While  they 
found  numerous  bacteria  in  the  vestibulum  naris,  cultures  made  from 
mucus  obtained  from  the  interior  of  the  nose  usually  gave  a  negative 
result— sixty -four  out  of  seventy-six  remained  absolutely  sterile, 
while  in  seven  there  was  a  scanty  growth  only.  They  conclude  that 
while  microorganisms  are  occasionally  found  upon  the  Schneider- 
ian  membrane  they  are  not  numerous  and  are  often  entirely  absent; 
and  that  they  are  rarely  found  upon  the  pituitary  membrane.  Straus 
(1895)  has  examined  the  nasal  secretions  of  persons  associated  with 
tubercular  patients  for  the  purpose  of  ascertaining  if  the  tubercle  ba- 
cillus was  present.  The  presence  of  this  bacillus  was  demonstrated, 
by  inoculation  into  guinea-pigs,  in  nine  healthy  individuals  out  of 
twenty -nine  examined ;  two  of  these  were  physicians  and  six  were 
nurses. 

Very  extended  researches  have  been  made  with  reference  to  the 
bacteria  present  in  the  human  mouth,  which  show  that  numerous 
species  are  constantly  present  in  the  buccal  secretions  and  upon  the 
surface  of  the  moist  mucous  membrane.  Some  of  these  are  occa- 
sional and  accidental,  while  others  appear  to  have  their  normal  habi- 


662          BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

tat  in  the  mouth,  where  the  conditions  as  to  temperature,  moisture, 
and  presence  of  organic  pabulum  are  extremely  favorable  for  their 
development.  A  minute  drop  of  saliva  spread  upon  a  glass  slide, 
dried,  and  stained  with  one  of  the  aniline  colors,  will  always  be 
found  to  contain  an  immense  number  of  bacteria  of  various  forms. 
Some  of  these  are  attached  to  epithelial  cells  and  some  scattered  about 
singly  or  in  groups.  Among  those  seen  in  a  single  specimen  we  will 
usually  find  cocci  in  tetrads,  in  chains,  and  in  irregular  groups, 
bacilli  of  various  dimensions,  and  occasionally  spirilla.  According 
to  Prof.  Miller,  of  Berlin,  the  following  species  almost  invariably 
occur  in  every  mouth :  Leptothrix  innominata,  Bacillus  buccalismax- 
imus,  Leptothrix  buccalis  maxima,  lodococcus  vaginatus,  Spirillum 
sputigenum,  Spirocha3te  dentium.  All  of  these  fail  to  grow  in  ordi- 
nary culture  media.  Miller  has  made  extended  attempts  to  obtain 
cultures  by  varying  the  medium  used  and  attempting  to  imitate  as 
nearly  as  possible  the  natural  medium  in  which  they  are  found;  but 
his  attempts  have  been  unsuccessful,  or  nearly  so — "  only  line  cultures 
afforded  a  limited  growth,  but  the  colonies  never  developed  more 
than  fifteen  to  twenty  cells,  and  a  transference  to  a  second  plate 
proved  futile,  no  further  growth  taking  place." 

Up  to  the  year  1885  Miller  had  isolated  twenty-two  different 
species  of  bacteria  from  the  human  mouth.  Ten  of  these  were  cocci, 
five  short  bacilli,  six  long  bacilli,  and  one  a  spirillum.  Later  the 
same  author  cultivated  eight  additional  species.  Vignal  has  isolated 
and  described  seventeen  species  obtained  by  him  in  pure  cultures 
from  the  healthy  human  mouth;  most  of  these  are  bacilli,  and  Miller, 
who  found  micrococci  to  be  more  numerous,  supposes  the  difference 
in  results  to  be  due  to  the  fact  that  many  of  the  cocci  do  not  grow  in 
nutrient  gelatin,  which  was  the  medium  employed  by  Vignal.  In 
the  researches  of  the  last-named  author  the  following  species  were 
obtained  most  frequently,  in  the  order  given:  1.  Bacterium  termo. 
2.  Bacillus  e  (Bacillus  ulna  ?).  3.  Potato  bacillus.  4.  Coccus  a.  5. 
Bacillus  b.  6.  Bacillus  d.  7.  Bacillus  c  (Bacillus  alvei  ?).  8.  Bacil- 
lus subtilis.  9.  Staphylococcus  pyogenes  albus.  10.  Staphylococcus 
pyogenes  aureus. 

Among  the  species  above  enumerated  we  find  two  of  the  most 
common  pus  cocci,  Staphylococcus  albus  and  aureus.  but  no  mention 
is  made  of  another  important  pathogenic  micrococcus  which  is  fre- 
quently found  in  the  healthy  human  mouth,  viz.,  the  micrococcus  of 
sputum  septicaemia,  first  named  by  the  writer  Micrococcus  Pasteuri. 
This  does  not  grow  at  ordinary  temperatures,  and  consequently 
would  not  be  obtained  in  gelatin  plate  cultures.  Very  different  re- 
sults have  been  reported  by  different  observers  as  to  the  frequency 
with  which  the  pathogenic  cocci  are  found  in  the  buccal  cavity. 


AND    OF   EXPOSED    MUCOUS   MEMBRANES.  663 

Black  found  in  the  saliva  of  ten  healthy  individuals  the  Staphy- 
lococcus  pyogenes  aureus  seven  times,  Staphylococcus  pyogenes  al- 
bus  four  times,  and  Streptococcus  pyogenes  three  times.  On  the 
other  hand,  better  found  Staphylococcus  aureus  only  seven  times  in 
one  hundred  and  twenty-seven  individuals  examined.  Miller  also 
has  rarely  found  the  pus  cocci  in  the  mouths  of  healthy  persons. 
Streptococcus  pyogenes  was  not  found  by  Vignal  in  his  extended 
researches.  The  experiments  of  the  writer,  of  Vulpian,  Frankel, 
Netter,  Claxton,  and  others  show  that  the  micrococcus  which  in 
1885  I  named  Micrococcus  Pasteuri,  and  which  is  identical  with  the 
"  diplococcus  pneumonia  "  of  German  authors,  is  frequently  present 
in  the  healthy  human  mouth— now  called  Micrococcus  pneumonia 
crouposaa.  Netter  examined  the  saliva  of  one  hundred  and  sixty-five 
healthy  individuals  and  obtained  it  in  fifteen  per  cent  of  the  number 
examined. 

Another  pathogenic  micrococcus  which  is  frequently  present  in 
the  mouths  of  healthy  persons  is  the  Micrococcus  tetragenus  of  Koch. 
The  following  pathogenic  bacteria  have  also  been  isolated  and  de- 
scribed :  Bacillus  crassus  sputigenus  (Kreibohm),  Bacillus  salivarius 
septicus  (Biondi).  The  Streptococcus  septo-pysemicus  of  Biondi  is 
described  as  having  characters  identical  with  those  of  the  Strepto- 
coccus pyogenes  of  Rosenbach.  Two  other  pathogenic  species  de- 
scribed by  Biondi  were  each  found  in  a  single  case  only.  Miller 
has  described  the  following  pathogenic  species  isolated  and  studied 
by  him  :  Micrococcus  gingiva3  pyogenes,  Bacterium  gingiva3  pyo- 
genes, Bacillus  dentalis  viridans,  Bacillus  pulpaB  pyogenes. 

Rosen  thai  (1893)  examined  the  secretions  from  the  mouths  of 
fourteen  individuals  and  obtained  twenty-eight  different  bacteria;  of 
these  twenty-one  had  been  previously  described.  Five  species  be- 
lieved to  be  new  are  described  in  detail  by  Rosenthal,  viz. :  Sarcina 
viridis  flavescens,  Micrococcus  Reessii,  Micrococcus  ochraceus,  Dip- 
lococcus Hauseri,  Bacterium  cerasinum. 

Vignal  has  tested  a  considerable  number  of  microorganisms, 
obtained  by  him  in  his  cultures  from  the  healthy  human  mouth,  with 
reference  to  their  peptonizing  action  upon  various  kinds  of  food,  with 
the  idea  that  some  of  them  may  have  an  important  physiological 
function  of  this  kind.  Out  of  nineteen  species  he  found  ten  which, 
after  a  longer  or  shorter  time,  dissolved  fibrin,  nine  which  dissolved 
gluten,  ten  which  dissolved  casein,  and  five  which  dissolved  albumin ; 
nine  changed  lactose  into  lactic  acid,  seven  inverted  cane  sugar,  seven 
caused  the  fermentation  of  glucose,  and  seven  coagulated  milk. 

Sanarelli  (1891)  has  shown  that  normal  saliva  has  the  power 
of  destroying  the  vitality  of  a  limited  number  of  certain  patho- 
genic bacteria,  including  the  following  species:  Staphylococcus 
pyogenes  aureus,  Streptococcus  pyogenes,  Micrococcus  tetragenus 


6G4         BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

Bacillus  typhi  abdominalis,  Spirillum  cholerse  Asiatics.  When  to 
ten  cubic  centimetres  of  saliva,  sterilized  by  filtration  through  porce- 
lain, the  above-mentioned  pathogenic  bacteria  were  added  in  small 
numbers  by  means  of  a  platinum  needle  carried  over  from  a  pure 
culture,  no  development  occurred,  and  at  the  end  of  twenty-four 
hours  the  bacteria  introduced  were  incapable  of  growth  in  a  suitable 
medium.  But  when  this  amount  of  filtered  saliva  was  inoculated 
with  a  large  platinum  loop — an  ose — a  certain  number  of  the  bacteria 
survived,  and  at  the  end  of  three  or  four  days  an  abundant  develop- 
opinent  occurred.  At  first,  however,  the  number  of  living  cells  was 
considerably  diminished.  In  saliva  to  which  one  ose  of  a  culture  of 
Staphylococcus  aureus  was  added  thirteen  thousand  eight  hundred 
and  forty  colonies  developed  in  a  plate  made  immediately  after  inocu- 
lation, while  a  plate  made  at  the  end  of  twenty-four  hours  contained 
but  one  hundred  and  thirty-two  colonies,  and  one  at  the  end  of  forty- 
eight  hours  had  but  eight  colonies.  Subsequently  multiplication 
occurred,  and  a  plate  made  on  the  ninth  day  after  inoculation  con- 
tained so  many  colonies  that  they  could  not  be  counted. 

The  diphtheria  bacillus  was  not  destroyed  in  filtered  saliva,  but 
did  not  multiply  in  it.  On  the  other  hand,  it  proved  to  be  a  very 
favorable  medium  for  the  development  of  Micrococcus  pneumonise 
crouposa3. 

Mucus  from  the  surface  of  the  meatus  urinarius  of  man  and 
woman,  or  from  the  vagina,  will  always  be  found  to  contain  various 
bacteria  ;  but  the  bladder,  the  uterus,  and  Fallopian  tubes  in  healthy 
individuals  are  free  from  microorganisms. 

Winter  has  isolated  twenty-seven  different  species  from  vaginal 
and  cervical  mucus,  and  reports  that  he  found  Staphylococcus  pyo- 
genes  albus  in  one-half  of  the  cases  examined.  A  streptococcus  was 
also  encountered  which  resembled  Streptococcus  pyogenes,  although 
not  positively  identified  with  it.  Samschin,  on  the  other  hand,  failed 
to  obtain  the  pus  cocci  in  vaginal  mucus  from  healthy  women. 

Donderlein,  Von  Ott,  and  others  have  carefully  examined  the 
lochial  discharge  with  reference  to  the  presence  of  bacteria.  The 
first-named  author  found  that  in  healthy  women  the  lochial  discharge 
obtained  from  the  uterus  was  free  from  germs,  but  when  collected 
from  the  vagina  various  microorganisms  were  obtained.  In  one  case 
in  which  some  fever  existed  Staphylococcus  pyogenes  aureus  was 
found  in  the  vagina,  while  the  discharge  from  the  uterus  was  free 
from  germs.  In  five  cases  of  puerperal  fever  Streptococcus  pyogenes 
was  obtained  in  the  lochial  discharge  from  the  uterus.  The  results 
of  Von  Ott  correspond  with  those  of  Donderlein.  Czerniewski,  in 
the  lochia  of  fifty-seven  healthy  women,  found  the  Streptococcus 
pyogenes  but  once,  while  in  the  lochial  discharge  of  fatal  cases  of 
puerperal  fever  it  was  always  present. 


AND   OF  EXPOSED   MUCOUS  MEMBRANES.  665 

Steffeck  (1892)  has  examined  the  vaginal  secretion  of  twenty-nine 
pregnant  females  who  had  not  been  subjected  to  digital  examina- 
tion, and  found  Staphylococcus  pyogenes  albus  in  nine,  Staphylo- 
coccus  pyogenes  aureus  in  three,  and  Streptococcus  pyogenes  in  one. 
These  results  indicate  that  puerperal  septicaemia  from  self-infection 
may  occur  in  exceptional  cases.  In  seventeen  of  the  twenty-nine 
cases  examined  none  of  these  pyogenic  micrococci  were  found. 

Hofmeister  (1894)  has  shown  that  bacteria  are  found  not  only 
upon  the  mucous  membrane  of  the  meatus  urinarius  in  man,  but 
that  they  may  usually  be  obtained  from  the  urethral  canal  at  a  depth 
of  eight  centimetres  or  more,  although  the  number  rapidly  diminishes 
in  the  deeper  portion  of  the  urethra. 

Walthard  (1895)  arrives  at  the  conclusion  that  while  in  pregnant 
females  bacteria  are  constantly  found  in  the  vagina  and  the  lower 
portion  of  the  cervical  canal,  they  are  absent  from  the  upper  part  of 
the  cervical  canal,  the  uterus,  and  the  tubes;  and  that  during  the 
puerperal  condition  the  uterine  cavity  is  preserved  from  spontaneous 
infection  per  vias  naturalis  by  the  plug  of  mucus  in  the  cervical 
canal.  In  the  vaginal  secretions  of  one  hundred  pregnant  women, 
who  had  not  been  subjected  to  a  digital  examination,  streptococci 
were  obtained  twenty-seven  times  in  cultures.  These  were  not  viru- 
lent, but,  according  to  Walthard,  these  saprophytic  streptococci  be- 
come virulent  when,  owing  to  a  diminished  resisting  power,  they  are 
enabled  to  invade  the  tissues  as  parasites. 

Kronig  (1894)  concludes  from  his  investigations  that  the  vaginal 
secretions  of  pregnant  women  are  usually  so  acid  that  Streptococcus 
pyogenes  could  not  multiply  in  them ;  also  that  when  the  secretion  is 
normal  it  is  almost  always  sterile. 

Doderlein  (1894)  insists  that  the  failure  of  Kronig  to  obtain  micro- 
organisms in  his  cultures  was  due  to  the  fact  that  suitable  media 
were  not  used ;  also  that  certain  bacilli  are  constantly  found  in  nor- 
mal, acid  vaginal  secretions,  and  that  in  the  pathological  secretions 
which  are  feebly  acid,  neutral,  or  in  some  cases  slightly  alkaline  a 
great  variety  of  bacteria  are  found,  including  Streptococcus  pyo- 
genes, as  demonstrated  by  himself  and  other  investigators.  In  a 
later  paper  (1894)  Kronig  reports  his  success  in  obtaining  cultures 
from  normal,  acid  vaginal  secretions  by  using  acid  media  and  by 
cultivating  under  anaerobic  conditions.  He  reports  also  that  patho- 
genic bacteria  (streptococci,  staphylococci,  and  Bacillus  pyocyaneus) 
introduced  into  the  vaginae  of  pregnant  women  lose  their  power  of 
reproduction  in  from  six  to  forty-eight  hours  (streptococci  did  not 
grow  after  six  hours).  In  a  still  later  communication  (1894)  Kronig 
reports  that  the  bacteria  present  in  the  vaginal  secretions  of  pregnant 

46 


GOO         BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

women  are  for  the  most  part  strictly  anaerobic  species,  and  that 
among  these  he  found  two  non-pathogenic  streptococci. 

Menge  (1894)  has  examined  the  vaginal  secretions  in  fifty  non- 
pregnant  women  who  had  been  in  bed  for  at  least  fourteen  days — 
after  laparotomy.  Microscopical  examination  showed  the  presence 
of  bacteria  in  all  cases,  but  in  only  six  cases  was  a  development  of 
colonies  obtained — upon  agar  plates;  in  one  case  Streptococcus  pyo- 
genes  was  present.  Menge  concludes  from  his  investigations  that 
spontaneous  infection  during  childbirth  cannot  occur,  and  that  with 
the  exception  of  the  gonococcus  the  known  pathogenic  bacteria  can- 
riot  multiply  in  the  cervical  canal. 

Gawronsky  (1894)  has  examined  the  secretions  from  the  healthy 
urethra  in  sixty-two  women,  most  of  whom  were  under  treatment 
for  uterine  disease  or  displacement.  The  material  for  his  cultures 
was  obtained  by  means  of  a  platinum  loop,  introduced  through  a 
glass  cylinder,  at  a  distance  of  one  or  one  and  one-half  centimetres 
from  the  external  orifice  of  the  urethra.  In  fifteen  out  of  the  sixty- 
two  cases  examined  a  positive  result  was  obtained,  as  follows:  In 
three  cases  Streptococcus  pyogenes,  in  eight  Staphylococcus  pyogenes 
aureus,  in  one  Staphylococcus  pyogenes  albus,  in  two  Bacillus  coli 
communis,  in  one  Bacterium  tholoideum  of  Gessner. 

The  following  species  have  been  obtained  from  the  nasal  and 
buccal  secretions  : 

FROM  THE  NOSE. 

Non-pathogenic. — Micrococcus  nasalis  (Hajek),  Diplococcus  coryzas 
(Hajek),  Micrococcus  albus  liquefaciens  (Von  Besser),  Micrococcus  cumu- 
latus  tenuis  (Von  Besser),  Micrococcus  tetragenus  subflavus  (Von  Besser), 
Diplococcus  fluorescens  foatidus  (Klamann),  Micrococcus  fcetidus  (Klamann), 
Vibrio  nasalis  (Weibel),  Bacillus  striatus  flavus  (Von  Besser),  Bacillus 
striatus  albus  (Von  Besser). 

Pathogenic. — Staphylococcus  pyogenes  aureus,  Staphylococcus  pyogenes 
albus,  Streptococcus  pyogenes,  Bacillus  of  Friedlander,  Bacillus  of  rhino- 
scleroma  (?),  Bacillus  fcetidus  ozseiiae  (Hajek),  Bacillus  mallei  (Loffler),  Ba- 
cillus smaragdinus  foetidus  (Reimann,). 

FROM  THE  MOUTH. 
Non-pathogenic. — Micrococcus  roseus  (Eisen berg),  Micrococcus  A,  B,  C, 


mann),  Bacillus  mesentericus  vulgatus,  Bacillus  subtilis,  Bacillus  a,  6,  <*.  </, 
e»/»  Qt  h,  *»  andj[of  Vignal,  Bacillus  subtilis  similis,  Bacillus  radicifonnis 
(Eisenberg),  Bacillus  luteus,  Bacillus  fluorescens  non-liquefaciens,  Bacillus 
ruber,  Bacillus  viridiflavus,  Proteus  Zenkeri,  Bacillus  G,  H,  I,  J,  K,  L,  M, 
N,  and  Vibrio  O  and  P  of  Podbielskij,  Vibrio  viridans  (Miller),  Micrococcus 
nexifer  (Miller),  lodococcus  magnus  (Miller),  Ascococcus  buccalis  (Miller), 
Bacillus  fuscans  (Miller). 

Pathogenic. — Staphylococcus  pyogenes  albus,  Staphylococcus  pyogenes 
aureus,  Staphylococcus  salivarius  septicus  (Biondi),  Streptococcus  pyogenes, 
Micrococcus  salivarius  septicus  (Biondi),  Micrococcus  tetragenus  (Gaffky), 


AND    OF   EXPOSED   MUCOUS   MEMBRANES.  667 

Micrococcus  gingivse  pyogenes  (Miller),  Streptococcus  septo-pyaemicus  (Bi- 
ondi),  Streptococcus  articulorum  (Loffler),  Micrococcus  of  Manfredi,  Micro- 
coccus  pneuinoniae  crouposae — ' '  Micrococcus  Pasteuri "  (Sternberg) ;  Bacillus 
diphtherias  (Loffler),  Bacillus  tuberculosis  (Koch),  Bacillus  of  Friedlander, 
Bacillus  bronchitidae  putridae  (Lumnitzer),  Bacillus  septicaemias  haemorrha- 
gicae,  Bacillus  gingivas  pyogenes  (Miller),  Bacillus  pulpae  pyogenes  (Miller), 
Bacillus  dentalis  viridans  (Miller),  Bacillus  crassus  sputigenus  (Kreibohm), 
Bacillus  saprogenes  No.  1  (Rosenbach),  Bacillus  pneumoniae  agilis  (Schou), 
Bacillus  pneumoniae  of  Klein,  Bacillus  pneumosepticus  (Babes). 


V. 
BACTERIA  OF  THE  STOMACH  AND   INTESTINE. 

,  As  the  secretions  of  the  mouth  contain  numerous  bacteria,  these 
must  constantly  find  their  way  to  the  stomach,  but  conditions  are 
not  favorable  for  their  development  when  the  stomach  is  in  a  healthy 
state  and  its  secretions  normal.  Under  certain  circumstances,  how- 
ever, there  may  be  an  abundant  development  in  the  stomach  of  spe- 
cies which  give  rise  to  various  fermentations,  and  no  doubt  dyspep- 
tic symptoms  are  frequently  due  to  this  cause.  In  the  present 
section  we  are,  however,  only  concerned  with  the  bacteria  of  the 
healthy  stomach.  Most  of  these,  we  think,  are  to  be  considered  as 
only  temporarily  and  accidentally  present  in  this  viscus  as  the  result 
of  the  swallowing  of  the  buccal  secretions  and  of  food  and  drink  con- 
taining them. 

The  experiments  of  Straus  and  Wurtz  and  of  others  show  that 
normal  gastric  juice  possesses  decided  germicidal  power,  which  is 
due  to  the  free  hydrochloric  acid  contained  in  it.  Hamburger  (1890) 
found  that  gastric  juice  containing  free  acid  is  almost  always  free 
from  living  microorganisms,  and  that  it  quickly  kills  the  cholera 
spirillum  and  the  typhoid  bacillus,  but  has  no  effect  upon  anthrax 
spores.  Straus  and  Wiirtz  found  that  the  cholera  spirillum  is  killed 
by  two  hours'  exposure  in  gastric  juice  obtained  from  dogs,  the 
typhoid  bacillus  in  two  to  three  hours,  anthrax  bacilli  in  fifteen  to 
twenty  minutes,  and  the  tubercle  bacillus  in  from  eighteen  to  thirty- 
six  hours.  The  experiments  of  Kurlow  and  Wagner,  made  with 
gastric  juice  obtained  from  the  stomach  of  healthy  men  by  means  of 
a  stomach  sound,  gave  the  following  results  :  Anthrax  bacilli  with- 
out spores  failed  to  grow  after  exposure  to  the  action  of  human  gas- 
tric juice  for  half  an  hour,  but  spores  were  not  destroyed  in  twenty- 
four  hours ;  the  typhoid  bacillus  was  killed  in  one  hour ;  the 
cholera  spirillum,  the  bacillus  of  glanders,  and  Bacillus  pyocyanus 
were  all  destroyed  at  the  end  of  half  an  hour  ;  the  pus  cocci  showed 
greater  resisting  power.  Certain  bacteria  have  a  greater  resisting 
power  for  acids  than  any  of  those  above  mentioned,  and  some  of  them 
may  consequently  pass  through  the  healthy  stomach  to  the  intestine 


BACTERIA   OF   THE    STOMACH   AND    INTESTINE.  669 

in  a  living  condition,  but  there  is  good  reason  to  believe  that  the 
spirillum  of  cholera  or  the  bacillus  of  anthrax  would  not.  On  the 
other  hand,  the  tubercle  bacillus  and  the  spores  of  other  bacilli  can, 
no  doubt,  pass  through  the  stomach  to  the  intestine  without  losing 
their  vitality. 

Of  nineteen  species  isolated  by  Vignal  in  his  cultures  from  the 
healthy  human  mouth,  the  greater  number  resisted  the  action  of  the 
gastric  juice  for  more  than  an  hour,  and  six  species  which  did  not 
form  spores  were  found  to  retain  their  vitality  in  gastric  juice  for 
more  than  twenty-four  hours. 

In  making  a  bacteriological  analysis  of  the  contents  of  the  healthy 
stomach  the  more  resistant  microorganisms  and  those  which  form 
spores  will  naturally  be  found  in  greater  or  less  numbers,  inasmuch 
as  some  of  them  are  likely  to  be  present  in  food  and  water  ingested. 

Van  Puteren  (1888)  obtained  a  variety  of  microorganisms  in  very 
considerable  numbers  from  the  stomachs  of  infants  fed  upon  un- 
sterilized  cow's  milk,  but  in  healthy  nursing  infants  the  number  was 
much  smaller,  especially  when  the  mouth  was  washed  out  with  dis- 
tilled water  immediately  before  and  after  nursing.  In  18  per  cent 
of  the  cases  no  microorganisms  were  found  under  these  circum- 
stances, and  in  41  per  cent  the  number  fell  below  one  thousand  per 
cubic  centimetre.  Among  the  nursing  infants  examined  (eighty- 
five)  the  following  species  were  most  numerous  :  Monilia  Candida, 
Bacillus  lactis  aerogenes,  a  non-liquefying  coccus,  Staphylococcus 
pyogenes  aureus,  Bacillus  subtilis.  In  infants  fed  upon  cow's  milk 
(eleven)  Bacillus  lactis  aerogenes  was  present  in  45.4  per  cent  of 
the  cases,  and  Staphylococcus  pyogenes  aureus  in  27.2  per  cent,  non- 
liquefying  cocci  in  54. 4  per  cent,  liquefying  cocci  in  72. 7  per  cent, 
Bacillus  subtilis  in  36.3  per  cent,  and  Bacillus  butyricus  (Hueppe) 
in  all  of  the  cases  ;  next  to  these  Bacillus  flavescens  liquefaciens 
was  the  most  abundant.  The  author  named  reaches  the  conclusion 
that  no  species  is  constant  and  that  the  presence  of  those  found  de- 
pends upon  accidental  circumstances. 

Abelous  (1889)  found  in  his  own  stomach,  washed  out  while  fast- 
ing, a  considerable  number  of  species  of  bacteria,  viz.  :  Sarcina 
ventriculi,  Bacillus  pyocyaneus,  Bacillus  lactis  aerogenes,  Bacillus 
subtilis,  Bacillus  mycoides,  Bacillus  amylobacter,  Vibrio  rugula, 
and  eight  other  undescribed  bacilli  and  one  coccus.  All  of  these 
microorganisms  were  able  to  resist  the  action  of  hydrochloric  acid 
in  the  proportion  of  1.7  grammes  in  1,000  grammes  of  water. 
Several  were  found  to  be  facultative  anaerobics. 

The  action  of  the  bacteria  isolated  by  him  was  tested  by  Abelous 
upon  various  alimentary  substances.  The  time  required  to  effect 
changes,  such  as  the  digestion  of  fibrin,  the  changing  of  starch 


670  BACTERIA   OF   THE   STOMACH   AND   INTESTINE. 

into  glucose,  etc.,  was  found  to  be  so  long  that  there  was  no  reason 
to  suppose  that  any  one  of  the  microorganisms  tested  was  con- 
cerned in  ordinary  stomach  digestion. 

In  the  intestine  conditions  are  favorable  for  the  development  of 
many  species  of  saprophytic  bacteria,  and  the  smallest  quantity  of 
excrementitious  material  from  the  bowels,  spread  upon  a  glass  slide 
and  stained  with  one  of  the  aniline  colors,  will  be  found  to  contain 
a  multitude  of  microorganisms  of  this  class,  of  various  forms. 
Among  these  are  certain  species  which  have  their  normal  habitat  in 
the  intestine,  and  which  may  always  be  obtained  in  cultures  from 
this  source,  while  others,  having  been  present  in  food  or  water  in- 
gested, and  having  escaped  destruction  in  the  acid  juices  of  the 
stomach,  are  accidentally  and  temporarily  present.  These  latter 
may  or  may  not  increase  in  the  organic  pabulum  which  abounds  in 
the  intestine,  according  as  the  conditions  are  favorable  or  otherwise. 
The  strictly  aerobic  bacteria  could  not  multiply  because  of  the  ab- 
sence of  oxygen,  and  the  species  encountered  are  for  the  most  part 
anaerobics  or  facultative  anaerobics.  The  Bacillus  coli  communis 
of  Escherich,  which  is  the  most  constant  and  abundant  species  found 
in  the  intestine  of  man  and  of  certain  of  the  lower  animals,  is  a  facul- 
tative anaerobic,  which  grows  readily  in  the  ordinary  culture  media, 
either  in  the  presence  of  oxygen  or  in  an  atmosphere  of  hydrogen. 
But  certain  other  bacteria  of  the  intestine  are  strictly  anaerobic  and 
do  not  grow  readily  in  the  media  commonly  employed  by  bacteri- 
ologists. 

Escherich  has  shown  that  in  new-born  infants  the  meconium  is 
free  from  bacteria.  At  the  end  of  twelve  to  eighteen  hours  after 
birth  bacteria  appear  in  the  alvine  discharges,  and  the  number  is 
already  considerable  at  the  expiration  of  the  first  twenty-four  hours 
of  independent  existence.  The  species  first  found  are  cocci  and  yeast 
cells  which  no  doubt  come  from  the  atmosphere,  having  been  de- 
posited upon  the  moist  mucous  membrane  of  the  mouth  and  swal- 
lowed with  the  buccal  secretions.  When  the  meconium  is  replaced 
by  "  milk  faeces "  these  contain  in  large  numbers  the  Bacillus  coli 
communis,  heretofore  spoken  of  as  the  most  common  species  found  in 
the  intestine  of  adults.  Another  species  associated  with  this,  but 
not  so  abundant,  is  the  Bacillus  lactis  aerogenes  of  Escherich. 
( )ther  bacilli  and  cocci  are  found  occasionally  in  smaller  numbers. 
These  bacilli  do  not  liquefy  gelatin,  and,  as  a  rule,  the  microor- 
ganisms found  in  the  alvine  discharges  of  healthy  persons  are  non- 
liquefying  bacteria.  Escherich's  researches  led  him  to  the  conclu- 
sion that  the  Bacillus  lactis  aerogenes  is  constantly  present  in  the 
small  intestine  of  milk-fed  children  as  the  most  prominent  species, 
and  that  its  multiplication  there  is  favored  by  the  presence  of  milk 


BACTERIA   OF   THE   STOMACH   AND   INTESTINE.  671 

sugar,  and  that  Bacillus  coli  communis  finds  the  most  favorable 
conditions  for  its  growth  in  the  large  intestine. 

Brieger,  in  1884,  isolated  from  faeces  and  carefully  studied  two 
bacilli,  one  of  which  has  since  been  called  by  his  name.  This  is  a 
non-liquefying  bacillus  which  is  very  pathogenic  for  guinea-pigs, 
and  which  in  its  morphology  and  characters  of  growth  closely  re- 
sembles the  Bacillus  coli  communis  of  Escherich.  Indeed,  a  num- 
ber of  non-liquefying  bacilli,  differing  but  slightly  in  their  morpho- 
logical and  biological  characters,  have  been  obtained  by  various 
investigators  from  the  alimentary  canal  of  man  and  the  lower  ani- 
mals, and  it  is  still  a  question  whether  they  are  to  be  regarded  as 
distinct  species  or  as  varieties  of  the  "colon  bacillus  "  of  Escherich. 
The  bacillus  obtained  by  Emmerich  from  cholera  cadavers  in  Na- 
ples belongs  to  this  group,  and,  if  not  identical  with  the  colon  bacil- 
lus, resembles  it  so  closely  that  its  differentiation  is  extremely  diffi- 
cult. Brieger's  bacillus  forms  propionic  acid  in  solutions  containing 
grape  sugar.  A  second  bacillus  obtained  by  him  from  the  same 
source  resembles  the  "  pneumococcus "  of  Friedlander  ;  this  causes 
the  fermentation  of  saccharine  solutions,  with  production  of  ethyl 
alcohol. 

Bienstock  (1883)  isolated  four  species  of  bacilli  from  normal  faeces, 
two  of  which  are  comparatively  large  and  resemble  Bacillus  sub- 
tilis  in  their  morphology  and  in  the  formation  of  spores.  A  third 
species  is  described  as  an  extremely  slender  pathogenic  bacillus,  re- 
sembling the  bacillus  of  mouse  septicaemia.  The  fourth  species  is  an 
actively  motile  bacillus  which  forms  end  spores,  causing  the  rods  to 
have  the  form  of  a  drumstick.  This  is  said  to  cause  the  decomposi- 
tion of  albumin,  with  production  of  ammonia  and  carbon  dioxide. 
Later  researches  do  not  sustain  Bienstock's  conclusion  that  the  ba- 
cilli described  by  him  are  the  principal  forms  found  in  normal  faeces. 

Among  the  species  encountered  by  Escherich,  in  addition  to  those 
mentioned  above  (Bacillus  coli  communis  and  Bacillus  lactis  aero- 
genes),  are  the  following :  Proteus  vulgaris,  found  three  times  in 
meconium,  and  constantly  in  the  faeces  of  dogs  fed  upon  flesh  ;  Strep- 
tococcus coli  gracilis,  found  in  meconium,  but  not  during  the  period 
of  nursing,  is  constantly  present  in  the  intestine  when  a  flesh  diet  is 
employed. 

The  intestine  of  carnivorous  and  omnivorous  animals  contains  a 
greater  number  of  bacteria  than  that  of  the  herbivora,  and  in  the 
large  intestine  they  are  far  more  numerous  than  in  the  small  intes- 
tine (De  Giaxa).  Sucksdorf  has  enumerated  the  colonies  developing 
from  one  milligramme  of  faeces  from  individuals  on  mixed  diet.  He 
obtained  an  average  of  380,000  from  a  series  of  observations  in  which 
the  maximum  was  2,300,000  and  the  minimum  25,000. 


672  BACTERIA   OF   THE   STOMACH   AND   INTESTINE. 

The  constant  presence  of  certain  species  of  bacteria  in  the  intes- 
tine of  man  and  the  lower  animals  has  led  to  the  supposition  that 
they  may  serve  a  useful  purpose,  or  perhaps  even  have  an  essential 
physiological  role  in  connection  with  intestinal  digestion.  While 
this  question  has  not  been  definitely  settled,  the  experiments  of 
Vallin,  Abelous,  and  others  have  thrown  some  light  upon  it,  and  a 
recent  experiment  by  Nuttall  and  Thierf elder  (1895)  has  considerable 
importance  as  bearing  upon  its  solution.  The  experiment  consisted 
in  removing  a  foetus  from  a  pregnant  guinea-pig  by  Cassarean  sec- 
tion, placing  it  under  conditions  which  protected  it  from  the  micro- 
organisms present  in  the  atmosphere,  and  feeding  it  upon  sterilized 
milk.  Great  technical  skill  was  shown  in  carrying  out  this  experi- 
ment for  a  period  of  eight  days,  during  which  time  the  little  animal 
was  kept  in  a  sterilized  atmosphere  and  was  fed  every  hour  day  and 
night.  At  the  end  of  this  time  it  had  consumed  over  three  hundred 
and  thirty  cubic  centimetres  of  sterilized  milk,  and  was  as  active  and 
healthy  as  other  guinea-pigs  of  the  same  age.  It  was  now  killed,  and 
a  careful  bacteriological  examination  showed  that  the  discharges 
from  the  bowels  and  the  contents  of  the  intestine  were  entirely  sterile. 

ADDITIONAL   NOTES   UPON   BACTERIA  OF   THE   STOMACH   AND 

INTESTINE. 

Oppler  (1894)  has  examined  material,  obtained  in  the  early  morning-,  from 
the  stomach  of  persons  suffering  from  indigestion,  and  found  nearly  always 
numerous  masses  of  sarcinae.  Five  different  species  were  obtained  from  this 
source,  which  were  distinguished  by  the  following  characters  :  No.  1,  colo- 
nies sulphur  yellow ;  No.  2,  colonies  greenish  yellow  ;  No.  3,  colonies  white ; 
No.  4,  colonies  white,  does  not  liquefy  gelatin  ;  No.  5,  colonies  orange  yel- 
low. Nos.  1  and  3  were  most  frequently  encountered. 

Kauff  mami  (1895)  in  a  carefully  studied  case  of  chronic  dyspepsia  obtained 
from  the  contents  of  the  stomach  in  the  morning  before  breakfast,  and  after 
a  test  meal,  the  following  bacteria  :  Yellow  sarcina,  Micrococcusaurantiacus, 
Staphylococcus  cereus  albus.  Bacillus  subtilis,  Bacillus  ramosus,  "  a  large 
thick  oacillus,"  u  a  short  bacillus  resembling  Bacillus  coli  comnmnis."  The 
last-mentioned  bacillus  was  found  in  large  numbers,  and  Kauffmann  suggots 
that  it  may  have  been  the  cause  of  the  fermentation  in  the  digestive  tract 
which  caused  the  unpleasant  symptoms  in  the  case  under  investigation. 

Macfayden  (1887)  and  Gillespie  (1893)  have  also  obtained  a  bacillus  from 
the  stomach  which  appears  to  be  identical  with  Bacillus  coli  communis.  In 
the  researches  of  Gillespie  it  was  obtained  from  a  patient  with  dilatation  of 
the  stomach  who  suffered  from  flatulence,  etc.  In  all,  twenty-four  different 
microorganisms  were  obtained  by  Gillespie  from  the  contents  of  the  stomach 
of  different  individuals.  This  number  includes  three  species  of  saccha- 
romyces  and  a  mucor.  Among  the  conclusions  reached  by  Gillespie  are  the 
following  : 

"14.  Although  bacteria  are  of  no  aid  to  peptic  digestion,  and  a  hindrance 
to  the  pancreatic  ferment  if  in  quantity  in  the  duodenum,  they  still  are  of 
great  use  in  the  small  intestine,  wnere  they  control  putrefaction.  This  seems 
paradoxical  :  microorganisms  obstructing  microorganisms  but  assisting  diges- 
tion. It  seems,  however,  to  be  true.  The  organisms  which  most  easily 
pass  tne  searching  examination  of  the  stomach  are  those  which  give  rise  by 


BACTERIA    OF   THE    STOMACH   AND    INTESTINE.  673 

their  growth  to  the  fatty  acids,  as  they  are  the  most  resistant  to  the  action  of 
acids.  Their  products  in  the  small  intestine  are  sufficient  to  keep  the  con- 
tents of  that  viscus  acid,  and  they  thereby  prevent  or  control  putrefaction. 
In  the  large  intestine  the  secretion  is  so  alkaline  that  the  putrefactive  organ- 
isms reassert  themselves. 

"  15.  Increased  putrefaction  in  the  intestinal  canal  may  therefore  be  due, 
in  some  cases,  either  to  insufficient  mortality  among  the  putrefactive  organ- 
isms in  the  stomach,  or  to  too  great  mortality  among  the  acid-forming  bac- 
teria and  yeasts. 

"16.  The  lactic  acid  which  appears  during  the  first  stages  of  digestion  is 
due  to  the  action  of  organisms. 

"17.  The  lactic,  acetic,  butyric,  and  succinic  acids  found  in  gastroectasis 
are  due  also  to  organisms  which  luxuriate  in  the  too  stationary  contents. 
The  marsh  gas,  the  Brennender-gas  of  the  Germans,  is  probably  due  to  the 
same  cause  ;  in  the  only  case  of  this  character  with  which  I  have  had  the 
good  fortune  to  meet  no  material  for  examination  could  be  obtained." 

The  following  species  have  been  isolated  from  faeces  and  the  con- 
tents of  the  intestine  of  cadavers  : 

Non-pathogenic. — Streptococcus  coli  gracilis  (Escherich),  Micrococcus 
aerogenes  (Miller),  Micrococcus  tetragenus  versatilis  (Sternberg),  Micrococ- 
cus ovalis  (Escherich),  "Yellow  liquefying  staphylococcus"  (Escherich), 
"  Porzellancoccus  "  (Escherich),  Bacillus  subtilis,  Bacillus  aerogenes  (Miller), 
Bacterium  aerogenes  (Miller),  Bacillus  lactis  erythrogenes  (Hueppe),  Clpstri- 
dium  fostidum  (Liborius),  Bacillus  muscoides(Liborius),  Bacillus  putrificus 
coli  (Bienstock),  Bacillus  subtilis  similis  I.  and  II.  (Bienstock),  Bacillus 
Zopfii,  Bacillus  liquefaciens  communis  (Sternberg),  Bacillus  in testinus  lique- 
faciens  (Sternberg),  Bacillus  intestinus  motilis  (Sternberg),  Bacillus  fluores- 
cens  liquefaciens  (Fliigge),  "Colorless  fluorescent  liquefying  bacillus" 
(Escherich),  "Yellow  liquefying  bacillus"  (Escherich),  Bacillus  mesenteri- 
cus  vulgatus,  Bacilli  of  Booker,  A  to  T,  first  series ;  a  to  s,  second  series ; 
Bacilli  of  Jeffries  A  to  Z,  and  a,  /?. 

Pathogenic. — Staphylococcus  pyogenes  aureus,  Bacillus  typhi  abdo- 
minalis,  Bacillus  septicaemias  haemorrhagicae,  Bacillus  of  Belfanti  and  Pas- 
carola,  Bacillus  enteritidis  (Gartner),  Bacillus  of  Lesage,  Bacillus  pseudo- 
murisepticus  (Bienstock),  Bacillus  coli  communis  (Escherich),  Bacillus  lactis 
aerogenes  (Escherich),  Bacillus  cavicida  (Brieger),  Bacillus  of  Emmerich, 
Bacillus  coprogenesfcetidus(Schottelius),  Bacillus  of  Utpadel,  Bacillus  leporis 
lethalis  (Sternberg),  Bacillus  acidiformans  (Sternberg),  Bacillus  cuniculicida 
Havaniensis  (Sternberg),  Bacillus  cadaveris  (Sternberg),  Bacillus  cavicida 
Havaniensis  (Sternberg),  Proteus  vulgaris  (Hauser),  Bacillus  tuberculosis, 
Spirillum  cholerae  Asiaticee  Spirillum  of  Finkler  and  Prior. 


VI. 

BACTERIA  OF    CADAVERS   AND    OF    PUTREFYING 
MATERIAL  FROM  VARIOUS  SOURCES. 

THE  putrefactive  changes  which  occur  so  promptly  in  cadavers, 
when  temperature  conditions  are  favorable,  result  chiefly  from  post- 
mortem invasion  of  the  tissues  by  bacteria  contained  in  the  alimen- 
tary canal.  But  it  is  probable  that  under  certain  circumstances 
microorganisms  from  the  intestine  may  find  their  way  into  the  cir- 
culation during  the  last  hours  of  life,  and  that  the  very  prompt  putre- 
factive changes  in  certain  infectious  diseases  in  which  the  intestine 
is  more  or  less  involved  are  due  to  this  fact.  The  writer  has  made 
numerous  experiments  in  which  a  portion  of  liver  or  kidney  re- 
moved from  the  cadaver  at  an  autopsy  made  soon  after  death — one 
to  six  hours — has  been  enveloped  in  an  antiseptic  wrapping  and  kept 
for  forty-eight  hours  at  a  temperature  of  25°  to  30°  C.  In  every  in- 
stance there  has  been  an  abundant  development  of  bacteria,  although 
as  a  rule  none  were  obtained  from  the  same  material  immediately  after 
the  removal  of  the  organ  from  the  body.  This  shows  that  a  few 
scattered  bacteria  were  present.  The  same  result  was  obtained  in 
cases  of  sudden  death  from  accident,  as  from  portions  of  liver  or 
kidney  removed  from  the  bodies  of  persons  dying  of  yellow  fever, 
tuberculosis,  and  other  diseases. 

Numerous  researches  show  that  the  blood  of  healthy  men  and 
animals  is  free  from  bacteria,  and  that  saprophytic  bacteria  injected 
into  a  vein  soon  disappear  from  the  circulation ;  and  recent  experi- 
ments show  that  blood  serum  has  decided  germicidal  power.  But  in 
spite  of  this  fact  the  experiments  of  Wyssokowitsch  show  that  cer- 
tain bacteria  injected  into  the  circulation  may  ba  deposited  in  the 
liver,  the  spleen,  and  the  marrow  of  the  bones,  and  there  retain  their 
vitality  for  a  considerable  time.  The  spores  of  Bacillus  subtilis  were 
found  by  the  observer  named  to  preserve  their  vitality  in  the  liver  or 
spleen  of  animals  into  which  they  had  been  injected,  for  a  period  of 
two  or  three  months.  In  the  writer's  experiments  the  microorgan- 
isms which  first  developed  in  fragments  of  liver  preserved  in  an  an- 
tiseptic wrapping  were  certain  large  anaerobic  bacilli,  and  especially 


BACTERIA  OF  CADAVERS  AND   OF  PUTREFYING  MATERIAL.      675 

my  Bacillus  cadaveris,  together  with  the  Bacillus  coli  communis 
of  Escherich,  my  Bacillus  hepaticus  fortuitus,  and  other  non-lique- 
fying bacilli  of  the  "colon  group." 

These  bacteria  did  not  give  rise  to  a  putrefactive  odor,  and  the 
fragment  of  liver  when  cut  into  had  a  fresh  appearance  and  a  very 
acid  reaction.  Later,  putrefactive  changes  occurred  and  Proteus 


FIG.  199.— Smear  preparation  from  liver  of  yellow-fever  cadaver,  kept  forty-eight  hours  in  an 
antiseptic  wrapping,    x  1,000.    From  a  photomicrograph.    (Sternberg.) 

vulgaris  and  other  putrefactive  bacteria  obtained  the  precedence. 
Evidently  all  of  these  species  must  have  been  present  in  the  liver  at 
the  time  it  was  removed  from  the  cadaver,  although  in  such  small 
numbers  that  they  were  rarely  seen  in  smear  preparations  or  ob- 
tained in  cultures  from  the  fresh  liver  tissue.  The  appearance  of  a 
smear  preparation  from  the  interior  of  a  fragment  preserved  for 
forty-eight  hours  in  an  antiseptic  wrapping  is  shown  in  Fig.  199. 

The  horribly  offensive  gases  which  are  given  off  from  dead  ani- 
mals in  a  state  of  putrefaction  appear  to  be  due  to  certain  large  an- 
aerobic bacilli  which  are  found  in  such  material, 
and  which  have  not  yet  been  thoroughly  studied 
owing  to  the  difficulty  of  cultivating  them  in  arti- 
ficial media ;  among  them  is  a  large  bacillus  with 
round  ends  which  forms  an  oval  spore  at  one  ex-  ^_ 

tremity  of  the  rather  long  rod.     This  the  writer  ^^  ^     y 

has  described  under  the  name  of  Bacillus  cada-  .  ^  ^^ 

veris  grandis,  Fig.  200.  %      p^  m 

In  the  interior  of  a  putrefying  mass  of  this  kind 

only  those  bacteria  are  found  which  are  able  to  grow  in  the  absence 
of  oxygen,  but  aerobic  saprophytes  may  multiply  upon  the  surface  of 


676      BACTERIA   OF  CADAVERS   AND   OF   PUTREFYING   MATERIAL. 

such  a  mass,  or  in  organic  liquids  to  which  the  air  has  free  access. 
Among  the  most  common  putrefactive  bacteria  are  the  Proteus  vul- 
garis,  Proteus  mirabilis,  and  Proteus  Zenkeri  of  Hauser.  Formerly 
the  minute  motile  bacteria  found  in  putrefying  animal  infusions,  etc., 
were  commonly  spoken  of  as  belonging  to  the  species  "  Bacterium 
termo,"  but  recent  researches  show  that  several  different  species  were 
included  under  this  name  by  those  whose  researches  were  made  be- 
fore the  introduction  of  Koch's  method  for  isolating  and  differentiat- 
ing microorganisms  of  this  class  by  the  use  of  solid  culture  media. 
The  different  species  of  Proteus  are  all  facultative  anaerobics.  They 
are  more  or  less  pathogenic,  and  according  to  Hauser  produce  a  chem- 
ical poison  which,  when  injected  into  small  animals,  causes  death  with 
all  of  the  symptoms  of  putrid  intoxication.  The  bacillus  of  mouse 
septicaemia,  which  was  first  obtained  by  Koch  from  a  putrefying  meat 
infusion,  is  also  pathogenic,  as  are  the  writer's  Bacillus  cadaveris 
and  various  other  anaerobic  bacteria  found  in  putrefying  material. 

Some  account  of  the  various  products  of  putrefaction  and  the 
microorganisms  concerned  in  their  production  will  be  found  in  Sec- 
tion IV.,  Part  Second,  of  the  present  volume. 


VII. 

BACTERIA  IN  ARTICLES  OF  FOOD. 

Milk  always  contains  bacteria,  unless  drawn  with  special  precau- 
tions into  a  sterilized  flask.  In  the  healthy  udder  of  the  cow  it  is 
sterile,  but  in  tuberculous  cows,  when  the  milk  glands  are  involved, 
tubercle  bacilli  may  find  their  way  into  the  milk  in  considerable 
numbers.  Ag  ordinarily  obtained  and  preserved,  milk  Is  greatly  ex- 
posed to  bacterial  contamination  from  various  sources  ;  desquamated 
cuticle  from  the  external  surface  of  the  udder  and  from  the  hands  of 
the  milker,  and  floating  particles  from  the  air  of  the  stable,  fall  into  it 
at  the  very  moment  it  is  drawn,  and  it  is  subsequently  contaminated 
by  bacteria  from  the  air,  and  from  water  used  in  washing  the  recep- 
tacles in  which  it  is  placed  or  added  to  it  by  the  thrifty  milkman. 
As  it  furnishes  an  excellent  nutrient  medium  for  many  of  the  bacteria 
which  are  thus  introduced  into  it,  under  favorable  conditions  of  tem- 
perature it  quickly  undergoes  changes  due  to  the  multiplication  in  it 
of  one  or  more  of  these  microorganisms.  The  acid  fermentation  and 
coagulation  of  the  casein  which  so  constantly  occurs  is  completely 
prevented  by  sterilizing  fresh  milk  in  flasks  provided  with  a  close- 
fitting  cork  or  cotton  air  filter.  Numerous  researches  have  been 
made  with  reference  to  the  microorganisms  found  in  milk  and  the 
various  fermentations  to  which  they  give  rise.  Naturally  a  great 
variety  of  species  will  be  found  in  an  extended  research,  but  all  are 
accidentally  present,  and  only  those  demand  special  attention  which 
produce  the  various  fermentations  of  this  fluid  commonly  encoun- 
tered, or  which  have  special  pathogenic  properties. 

Several  different  bacteria  produce  an  acid  fermentation  and  con- 
sequent coagulation  of  milk,  but  the  usual  agent  in  producing  this 
fermentation  is  the  Bacillus  acidi  lactici,  which  is  identical  with  the 
' '  ferment  lactique  "  of  Pasteur.  When  a  pure  culture  of  this  bacillus 
is  introduced  into  sterilized  milk  kept  at  a  temperature  of  25°  to  30°  C., 
coagulation  occurs  in  from  fifteen  to  twenty-four  hours.  A  uniform, 
gelatinous  mass  is  produced  which  does  not  subsequently  become 
dissolved  (Adametz).  Various  other  bacteria  produce  a  similar 
change,  including  a  number  of  common  water  bacteria,  several  spe- 


078  BACTERIA   IN   ARTICLES   OF   FOOD. 

cies  of  sarciua,  Staphylococcus  pyogenes  aureus,  and  other  pus  cocci. 
Usually  coagulation  is  due  to  the  combined  action  of  several  bacteria, 
among  which  Bacillus  acidi  lactici  is  apt  to  be  the  most  prominent. 

Other  bacteria  produce  coagulation  without  the  lactic  acid  fer- 
mentation. This  appears  to  be  due  to  the  formation  of  a  soluble 
ferment  which  acts  like  rennet,  causing  the  coagulation  of  milk 
which  has  a  neutral  or  slightly  alkaline  reaction.  The  coagu- 
lated casein  in  this  case  is  subsequently  redissolved.  The  bacteria 
which  produce  this  change  for  the  most  part  form  spores,  while  the 
lactic  acid  ferments  do  not.  If,  therefore,  milk  is  heated  nearly  to  the 
boiling  point  the  acid-forming  bacteria  will  be  destroyed  and  the 
spores  of  the  other  species  surviving  will  give  rise  to  coagulation 
without  the  production  of  lactic  acid.  Among  the  more  common 
microorganisms  of  this  group  are  the  Bacillus  butyricus  (Hueppe), 
Bacillus  mesentericus  vulgatus,  Loffler's  "  white  milk-bacillus/'  and 
the  bacilli  described  by  Duclaux  under  the  generic  name  of  Tyrothrix. 

Other  fermentations  are  produced  by  certain  chromogenic  bacteria, 
and  these,  as  a  rule,  are  not  as  harmless  from  a  sanitary  point  of  view 
as  those  above  referred  to.  Blue  milk  is  produced  by  the  presence  of 
Bacillus  cyanogenus,  yellow  milk  by  Bacillus  synxanthus  (Schroter) 
and  by  a  species  obtained  by  List  from  the  faeces  of  a  sheep  and 
another  found  by  Adametz  in  cheese.  The  well-known  Bacillus 
prodigiosus  produces  its  characteristic  red  pigment  when  present  in 
milk,  and  a  bluish-red  color  is  caused  by  Bacterium  lactis  erythrogenes 
(Hueppe). 

Viscous  fermentation  in  milk  is  produced  by  several  different  bac- 
teria, among  others  by  a  micrococcus  studied  by  Schmidt-Muhlheim, 
and  a  short  bacillus  isolated  by  Adametz — Bacillus  lactis  viscosus. 
Milk  which  has  undergone  this  change  is  unwholesome  as  food  ;  it 
is  recognized  by  the  long  filaments  which  are  produced  when  it  is 
touched  with  any  object  and  this  is  slowly  withdrawn. 

The  Caucasian  milk  ferment,  Bacillus  Caucasicus,  produces  a 
special  fermentation,  which  has  been  referred  to  in  Section  IV.,  Part 
Second  (page  132). 

Various  pathogenic  bacteria  have  occasionally  been  found  in  milk 
in  addition  to  the  tubercle  bacillus  already  referred  to.  Thus  Adam*  it  /, 
found  Staphylococcus  pyogenes  aureus  in  two  samples  which  had 
been  submitted  to  him  for  examination,  one  of  which  had  given  rise 
to  vomiting  and  diarrhoea.  Wyssokowitsch  cultivated  from  milk 
which  had,  been  standing  some  time  a  pathogenic  bacillus,  named  by 
him  Bacillus  oxytocus  perniciosus. 

The  special  microorganism  which  produces  the  poisonous  pto- 
maine called  by  Vaughaii  tyrotoxicoii  has  not  yet  been  isolated  ;  i  mi- 
do  we  know  the  exact  cause  of  scarlet  fever,  although  there  is  evi- 


BACTERIA   IN   ARTICLES   OF   FOOD.  679 

dence  that  this  disease  has  been  spread  by  the  use  of  contaminated 
milk,  as  have  also  diphtheria  and  typhoid  fever,  which  diseases  are 
due  to  bacilli  now  well  known.  As  the  cholera  spirillum  grows 
readily  in  milk,  this  disease  could  no  doubt  also  be  transmitted  in 
the  same  way. 

Sedgwick  and  Batchelder  (1892)  have  examined  a  large  number 
of  specimens  of  milk  obtained  in  Boston  and  vicinity,  for  the  purpose 
of  determining  the  number  of  bacteria  present.  They  found,  as  an 
average  of  several  trials,  that  milk  obtained  in  a  clean  stable,  from 
a  well-kept  cow,  by  milking  in  the  usual  way  into  a  sterilized  bottle, 
contained  530  bacteria  per  cubic  centimetre.  "  When,  however,  the 
milkman  used  the  ordinary  milk  pail  of  flaring  form,  seated  himself 
with  more  or  less  disturbance  of  the  bedding^  and  vigorously  shook 
the  udder  over  the  pail  during  the  usual  process  of  milking,*' the 
numbers  were  very  much  higher — on  an  average  30,500  per  cubic 
centimetre  immediately  after  milking.  The  average  of  fifteen  sam- 
ples taken  from  the  tables  of  persons  living  in  the  suburbs  of  Boston 
was  69,143  per  cubic  centimetre.  The  average  of  fifty-seven  sam- 
ples of  Boston  milk,  obtained  directly  from  the  milk  wagons  and 
plated  at  once,  was  2,355,500  per  cubic  centimetre.  The  average  of 
sixteen  samples  from  groceries  in  the  city  of  Boston  was  4,577,000 
per  cubic  centimetre. 

Prof.  Renk  found  in  the  milk  supply  of  Halle  from  G, 000, 000  to 
30,000,000  bacteria  per  cubic  centimetre — a  number  considerably  ex- 
ceeding that  usually  found  in  the  sewage  of  American  cities  (Sedg- 
wick). 

Cohii  and  Neumann  (1891)  have  shown  that  the  milk  of  healthy 
women  frequently  contains  bacteria,  and  that  Staphylococcus  pyo- 
genes  albus  is  the  species  most  frequently  found.  This  has  been 
confirmed  by  the  researches  of  Palleske  (1892),  Ringel  (1893)  and 
others.  The  last-mentioned  author  examined  the  milk  of  25  women 
recently  confined,  "12  of  whom  were  healthy  and  13  sick."  In  3 
cases  only  was  the  milk  sterile;  in  17  cases  Staphylococcus  pyogenes 
albus  was  found;  in  2  cases  Staphylococcus  pyogenes  aureus;  in  1 
case  both  albus  and  aureus ;  in  2  cases  Staphylococcus  pyogenes  albus 
and  Streptococcus  pyogenes.  The  streptococci  were  found  in  a  case 
of  mild  puerperal  fever  and  in  a  case  of  phlebitis. 

The  researches  of  Hirshberger  (1889),  of  Ernst  (1895),  and  of 
others  show  that  the  milk  of  tuberculous  cows  may  contain  tubercle 
bacilli  even  when  the  udder  of  the  animal  presents  no  evidence  of  a 
localized  tubercular  infection.  In  121  samples  of  milk  examined  by 
Ernst  from  36  different  cows,  19  gave  a  positive  result;  all  from  the 
milk  of  12  cows  in  which  no  evidence  of  tuberculosis  of  the  udder 
was  found  in  a  carefully  made  post-mortem  examination.  Among 


<J80  BACTERIA   IN  ARTICLES   OF  FOOD. 

the  bacteria  which  produce  unwholesome  changes  in  milk  are  several 
which  cause  it  to  become  viscous  or  soapy.  Among  these  we  may 
mention  Micrococcus  lactis  viscosus  of  Conn,  Micrococcus  Freuden- 
reichi  of  Guillebeau,  Bacillus  mesentericus  vulgatus,  and  Bacillus 
lactis  saponacei  of  Weighmann  and  Zirn.  A  considerable  number 
of  bacilli  are  known  which  give  rise  to  the  production  of  butyric 
acid  fermentation  in  milk  and  its  products.  Some  of  these  are  an- 
aerobic and  some  aerobic.  The  list  includes  the  following :  Bacillus 
butyricus  of  Prazmowski,  Bacillus  of  Liborius,  Bacillus  of  Botkin, 
Bacilli  of  Kadrowski. 

The  bitter  taste  which  milk  and  cheese  sometimes  acquire  is  due 
to  the  presence  of  special  bacterial  ferments ;  among  these  the  best 
known  are  an  aerobic,  liquefying  micrococcus  described  by  Conn,  a 
bacillus  described  by  Weighmann,  Micrococcus  casei  amari  and  Ba- 
cillus liquefaciens  lactis  amari  of  De  Freudenreich  (1895). 

In  fresh  butter  of  good  quality  comparatively  few  microorganisms 
are  found,  but  the  researches  of  Conn  show  that  the  characteristic 
and  agreeable  flavor  of  fresh  butter  is  due  to,  or  at  least  may  be  imi- 
tated by,  a  bacillus  which  is  concerned  in  the  ripening  of  cream 
under  normal  conditions.  Cultures  of  this  bacillus  (Bacillus  41  of 
Conn)  have  already  been  used  in  a  practical  way  by  butter  makers 
to  improve  the  flavor  of  their  product. 

Kreuger  (1890)  obtained  from  "cheesy  butter,"  having  a  disa- 
greeable odor,  various  bacteria.  Among  these  the  most  numer- 
ous were  an  oval  micrococcus  (Micrococcus  acidi  lactici,  Kreuger),  a 
slender  bacillus  resembling  Bacillus  fluorescens,  and  Bacillus  acidi 
lactici  of  Hueppe. 

Klecki  (1894)  has  isolated  from  rancid  butter  several  bacteria  not 
previously  described,  one  or  more  of  which  are  no  doubt  concerned 
in  the  production  of  the  rancid  taste  and  odor.  These  are  described 
under  the  following  names:  Bacillus  butyri,  Diplococcus  butyri,  a 
bacillus  resembling  lodococcus  vaginatus  of  Miller,  Tetracoccus 
butyri,  Bacillus  butyri  No.  2. 

Duclaux  (1887)  has  isolated  from  different  kinds  of  cheese  no  less 
than  eleven  different  species  of  bacteria,  which  he  believes  are  con- 
cerned in  the  "ripening  process."  Seven  of  these  are  aerobic  and 
four  anaerobic  species.  Adametz  (1889)  has  also  isolated  and  studied 
a  number  of  species  to  which  he  attributes  the  ripening  of  cheese. 

More  recently  Henrici  (1895)  has  studied  the  bacterial  flora  of 
cheese,  and  Marshal  (lS9f>)  has  shown  that  the  ripening  of  certain 
kinds  of  cheese  (fromages  mous)  is  probably  due  to  Oidium  lactis. 

Meats,  even  when  salted  and  smoked,  may  contain  living  patho- 
genic bacteria  which  were  present  prior  to  the  death  of  the  animal, 
and,  when  not  properly  preserved,  are  of  course  liable  to  be  invaded 
by  putrefactive  bacteria. 


BACTERIA   IN   ARTICLES   OF  FOOD.  681 

The  researches  of  Foster  (1889)  show  that  the  typhoid  bacillus, 
the  pus  cocci,  the  tubercle  bacillus,  and  the  bacillus  of  swine  plague 
resist  the  action  of  a  saturated  solution  of  salt  for  weeks  and  even  for 
months;  and  the  same  observer  found  that  the  ordinary  processes  of 
salting  and  smoking  did  not  destroy  the  tubercle  bacillus  in  the  flesh 
of  a  cow  which  had  succumbed  to  tuberculosis.  Beu  has  made  cul- 
tures from  a  large  number  of  specimens  of  fresh,  salted,  and  smoked 
meats  and  fish,  with  the  general  result  that  the  fresh  and  salted  meats 
were  found  to  contain  a  limited  number  of  bacteria  of  various  species, 
and  that  smoking  for  several  days  did  not  insure  the  destruction  of 
these  microorganisms.  In  specimens  of  sausage  six  days'  smoking 
did  not  destroy  a  liquefying  bacillus  which  was  present,  but  at  the 
end  of  six  weeks'  exposure  to  smoke  this  bacillus  no  longer  grew, 
while  a  non-liquefying  bacillus  present  in  the  same  specimen  had  not 
been  destroyed.  Fourteen  days'  smoking  sufficed  to  destroy  all  the 
microorganisms  in  a  specimen  of  bacon,  but  this  was  not  sufficient 
for  the  interior  portions  of  a  ham.  Among  the  bacteria  obtained  by 
Beu  from  smoked  meats  he  mentions  the  following  :  Staphylococcus 
cereus  albus,  Proteus  vulgaris,  Staphylococcus  pyogenes  aureus,  Ba- 
cillus liquefaciens  viridis,  etc.  The  number  of  colonies  which  de- 
veloped from  a  fragment,  the  size  of  a  mustard  seed  to  that  of  a  flax- 
seed,  taken  from  the  interior  of  the  meats  examined,  was  usually 
small;  and  the  presence  of  a  few  scattered  bacteria  of  these  common 
species  has  no  significance  from  a  sanitary  point  of  view,  except  as 
showing  that  pathogenic  bacteria  may  survive  in  infected  meats  after 
they  have  been  exposed  to  the  usual  processes  of  salting  and  smoking. 

Petri,  in  experiments  upon  the  bacillus  of  swine  plague  (Schweine- 
rothlauf),  arrived  at  the  following  results  : 

The  flesh  of  swine  which  died  of  this  disease  preserved  its  infec- 
tious properties  after  having  been  preserved  in  brine  for  several 
months,  and  the  same  flesh  salted  or  pickled  for  a  month  and  then 
smoked  for  fourteen  days  contained  the  rothlauf  bacillus  in  a  living 
and  unattenuated  condition.  At  the  end  of  three  months  virulent 
rothlauf  bacilli  were  still  obtained  from  a  smoked  ham,  but  they  were 
no  longer  found  at  the  end  of  six  months. 

Schrank  (1888)  has  made  cultures  from  both  the  albumin  and  the 
yolk  of  fresh  eggs,  and  finds  that  they  are  free  from  bacteria.  He 
thinks  that,  as  a  rule,  putrefactive  bacteria  obtain  access  to  the  inte- 
rior through  injured  places  in  the  shell,  although  exceptionally  the 
egg  may  be  infected  with  them  in  the  oviduct  of  the  fowl.  The  usual 
bacteria  concerned  in  the  putrefactive  changes  in  eggs  are,  according 
to  the  author  mentioned,  a  variety  of  Proteus  vulgaris  and  Bacillus 
fluorescens  putidus. 

Zorkendorfer  (1893)  has  cultivated  from  rotten  eggs  sixteen  dif- 
47 


G82  BACTERIA   IN   ARTICLES   OF  FOOD. 

ferent  bacilli,  all  of  which  are  described  in  detail  and  none  of  which 
were  found  to  correspond  with  previously  described  species  as  given 
in  Eisenberg's  Bacteriological  Diagnosis. 

Peters  (1889)  has  studied  the  flora  of  the  "sauerteig"  used  in 
Germany  as  yeast  for  leavening  bread.  In  addition  to  the  numerous 
cells  of  three  species  of  Saccharomyces,  he  finds  that  bacilli  are  present 
in  great  numbers,  as  shown  by  direct  microscopical  examination  and 
culture  experiments.  He  describes  five  species,  designated  Bacillus 
A,  B,  C,  D,  and  E,  which  are  commonly  present,  and  to  which  the 
acid  fermentation  of  the  dough  is  ascribed. 

In  Graham  bread  which  had  undergone  changes  making  it  unfit 
to  eat,  Kratschmer  and  Niemilowicz  have  found  the  Bacillus  mes- 
entericus  vulgatus,  which  appears  to  have  been  the  cause  of  the 
fermentation,  which  was  produced  in  bread  having  a  slightly  alka- 
line reaction  by  inoculating  it  with  a  pure  culture  of  this  bacillus. 
The  infected  bread  has  a  brownish  color,  a  peculiar  odor,  and  be- 
comes sticky  and  viscid. 

Uffelmann  (1890)  has  also  studied  the  bacteria  in  spoiled  rye 
bread,  and  obtained,  in  addition  to  common  mould  fungi,  Bacillus 
mesentericus  vulgatus  and  Bacillus  liodermus. 

Waldo  (1894)  has  shown  that  baking  does  not  sterilize  bread. 
This  was  to  have  been  expected  in  the  case  of  the  spores  of  bacilli, 
but  it  is  somewhat  surprising  to  find  that  two  species  of  Sarcina  and 
two  micrococci  survived  the  baking  process.  In  all  Waldo  obtained 
thirteen  species  of  bacteria  from  the  interior  of  sixty-two  loaves 
examined.  Bacillus  subtilis  and  allied  spore-forming  bacilli  were 
most  frequently  found,  and  the  statement  is  made  that  a  loaf  "  from 
a  low-class,  dirty  bakery  will  almost  invariably  contain  more  living 
bacteria  (or  their  spores)  than  one  from  a  good,  clean  bakery." 

Lehmann  (1894)  under  the  name  Bacillus  levans  has  described  a 
microorganism  which  closely  resembles  Bacillus  coli  communis.  This 
was  obtained  from  sour  dough,  and  was  believed  to  be  the  cause  of 
the  acid  fermentation  which  so  often  interferes  with  success  in  ob- 
taining sweet  and  wholesome  bread.  When  a  culture  of  this  bacil- 
lus was  added  to  flour  and  water,  without  the  addition  of  yeast,  an 
active  fermentation  occurred  and  the  dough  became  acid. 


INDEX. 


ABRIN,  experiments  of  Ehrlich,  270 

Abscesses,  etiology  of,  571 
formation  of,  222,  275,  276 
Micrococeus    pneumonise   croupos* 
in,  313 

Acetic  acid,  germicidal  action  of,  178 
production  of,  134 

Acetone,  antiseptic  value  of,  193 

Acids,  germicidal  action  of,  176-179 

Acne,  etiology  of,  571 

contagiosa   of   horses,   bacillus  of, 
508,  571 

Acute  rheumatism,  etiology  of,  610 

Ae"roscopes,  625 

Agar-agar,  43 

filtration  of,  44 

Agar-gelatin,  43 

Agitation,  germicidal  action  of,  15& 

Agua  coco,  as  a  culture  medium,  39 

Air,  bacteria  in,  623-635 
filter  of  cotton,  4 

Alcohol,  germie&al  value  of,  193 

Alexins,  273 

Alkalie%  germicidal  value  of,  179-181 

Alkaline  fermentation  of  urine,  137 

Alopecia,  etiology  of,  572 

Afum,  antiseptic  value  of,  184 

Aluminium  acetate,  antiseptic  value  of, 
184 

Ammonia,  germicidal  value  of,  180 
liberation  of,  140 
oxidation  of,  141 

Ammonium  carbonate,  antiseptic  value 

of,  184 

chloride,  antiseptic  value  of,  184 
fluosilicate,  antiseptic  value  of,  184 
sulphate,  antiseptic  value  of,  184 

Anaerobic  bacteria,  cultivation  of,  78-85 
bacilli,  531-548 
cultures,  79 

cultures,  Buchner's  method,  83 
cultures,  Esmarch's  method,  82 
cultures,  Franker s  method,  80 
cultures,  Liborius'  method,  83 
cultures,  Stern  berg's  method,  81 

Anaesthetics,  use  of,  97 

Angina,  etiology  of,  572 

Aniline  dyes,  germicidal  value  of,  193 
oil,  antiseptic  value  of,  194 


Anthrax,  etiology  of,  339 

bacillus,  discovery  of,  6 

bacillus,  toxic  products  of,  147 
Antiseptic  action,  conditions  governing, 
162 

value,  how  determined,  161 
Antiseptics,  comparative  value  of,  182, 
183 

definition  of,  160 

Antitoxin  of  diphtheria,  results  of  treat- 
ment, 386 

of  pneumonia,  268 

of  snake  venom,  269 

of  tetanus,  268 

of  tetanus,  from  milk,  271 
Antitoxins,  266-272 

do  not  dialyze,  273 
Appendicitis,  etiology  of,  573 
Arsenious  acid,  germicidal  value  of,  179 
Arthritis,  etiology  of,  573 
Arthrospores,  120 
Ascococcus,  17,  21 

Johnei,  327 

Aseptol,  germicidal  value  of,  193,  194 
Asporogenous  varieties,  126 
Attenuation  of  virulence,  127-129 

by  antiseptics,  128 

by  heat,  128 

BACILLI,  morphology  of,  22 
Bacillus,  generic  characters,  18 
A  of  Booker,  492 
acidiformans,  474 
of  acne  contagiosa  of  horses,  508 
aeTogenes  capsulatus,  514 
aeTogenes  meningitides,  529 
albus  cadaveris,  503 
alvei,  507 
anthracis,  340 

anthracis,  biological  characters,  341 
anthracis,  morphology,  340 
anthracis,  pathogenesis,  345 
anthracis,  spore  formation,  341,  343 
anthracis,  toxic  products,  345,  348 
anthracis,  varieties  of,  343 
of  Babes  and  Oprescu,  458 
of  Beck,  524 

of  Belfanti  and  Pascarola,  439 
bovis  morbificans,  525 


684 


INDEX. 


Bacillus  of  bubonic  plague,  520 

of  Bunzl  Federn,  520 

cadaveris,  541 

canalis  capsulatus,  506 

canal  is  parvus,  506 

of  Canon  and  Pielicke,  515 

capsulatus,  454 

capsulatus  mucosus,  512 

cavicida,  448 

cavicida  Havaniensis,  448 

of  Cazal  and  Vaillard,  458 

of  chancroid,  576 
"Bacillus"  of  cholera,  552 

of  cholera  in  ducks,  434 

cholerae  galliuarum,  429 
Bacillus  of  Chiari,  517 

chromo-aromaticus,  505 
.     coli  communis,  462-474 

coli  communis  in  bread,  682 

coli  communis  in  cystitis,  583 

coli    communis  in  peritonitis,  472, 
605 

coli  communis  in  pyelonephritis,  609 

coli  communis  from  stomach,  672 

coli  communis,  varieties  of,  466-472 

coprogenes  foetid  us,  497 

coprogenes  parvus,  447 

crassus  sputigenus,  449 

cuniculicida,  429 

cuniculicida  Havaniensis,  47 

of  Demme,  494 

dentalis  viridans,  501 

diphtheriae,  375-380 

diphtheria?,     biological    characters, 
375 

diphtheria,  branching  forms,  384 

diphtheriae  in  fauces  of  healthy  per- 
sons, 385 

diphtheria),  morphology  of,  375 

diphtheria!,  pathogenesis,  377 

diphtheria?,  persistence  after  recov- 
ery, 384,  385 

diphtheria;,  toxic  products  of,  379 

diphtheria,  varieties  of,  380 

diphtheriae  columbrarum,  382 

diphtheriae  vitulorum,  383 

of  Ducrey,  576 

of  von  Dungern,  519 

of  Emmerich  and  Weibel,  523 

endocarditidis  capsulatus,  493 

endocarditidis  griseus,  493 

cntcritidis.  452 

erysipelatos  suis,  442 

erysipelatos  suis,  pathogenesis,  445 

of  Eve  and  Lingard,  424 

of  Fiocca,  484 
f<rtidus  oznenii',  l'.'-"> 

of  fowl  cholera.  1*.".' 

of  Frettenseuche,  439 

of  Friedlandcr,  308 
galliiiarum,  45o 

of  Gerdcs,  5H7 

of  Qessncr,  505 


Bacillus  of  Gibier,  478 
gingivae  pyogenes,  501 
of  Gplasz,  425 
gracilis  cadaveris,  516 
of  grouse  disease,  452 
of    Guillebcau,   «,    l>,  and    c,    528, 

529 

of  Harris,  517 
heminecrobiophilus,  511 
of  hog  cholera,  434 
of  hog  cholera,  varieties  of,  438 
liominis  capsulatus,  450 
hydrophilus  fuscus,  455 
indigogenus,  507 
of  influenza,  388 
of  influenza,    biological  characters, 

389 

of  influenza,  pathogenesis,  389 
of  intestinal  diphtheria  in  rabbits, 

384 

of  Jeffries,  474 
of  Kartulis,  507 
of  Koubasoff,  426 
lactis  aeTogenes,  472 
of  Laser,  457 

of  Laser,  gas-forming  aerobic,  524 
leporis  lethalis,  478 
leprae,  414-416 
of  Lesage,  493 
of  Letzerich,  495 
of  Loeb,  460 
of  Lucet,  459 
of  Lumnitzer,  496 
of  Lustgarten,  422 
malaria',  596 
mallei,  417-422 
mallei,  pathogenesis,  419 
meningitidis  purulenta?,  504 
of  Mereshkowsky,  523 
monachae,  528 
mucosus  ozaenae,  518 
murisepticus,  442 
Neapolitanus,  462 
necrophorus,  497 
of  Nicolaier,  517 
of  Nocard,  426 
oadematis  afrobicus,  494 
oedematis  maligni,  537 
cedematis  maligni  No.  11    (Novy), 

545 

of  Okada.  509 

phlegmones  emphysematosa?,  547 
piscicidus,  525 
piscicidus  agilis,  522 
pneumonia',  308 
pneumosepticus,  454 
prodigiosus,    pigment    production, 

132 

pseudo-tuberculosis,  500,  530 
pseudo-tuberculosis  muriuin,  530 
pulpa1  pyogenes,  501 
of  purpura  ha'morrhagica  of  Babes, 

510 


INDEX. 


685 


Bacillus    of  purpura  haemorrhagica   of 
Kolb,  511 

of  purpura  haemorrhagica  of  Tizzoni 
and  Giovannini,  510 

pyocyaneus,  479-484 

pyocyaneus  ft  (P.  Ernst),  482 

pyocyaneus,  in  otitis  media,  482 

pyocyaneus,  pathogenesis,  480 

pyocyaneus,  pigments  produced  by, 
131 

pyocyaneus  pericarditidis,  482 

pyogenes  filiformis,  526 

pyogenes  fcetidus,  450 

pyogenes  soli,  512 

of  rabbit  septicaemia,  429 

of  rhinoscleroma,  425 

of  Rinderseuche,  440 

No.  I.  of  Roth,  509 

No.  II.  of  Roth,  509 

salivarius  septicus,  310 

sanguinis  typhi,  515 

of  Schimmelbusch,  495 

of  Schou,  497 

septicaemiae  haemorrhagicae,  429-434 

septicaemiae  haemorrrhagicae,  attenu- 
ation of,  432 

septicaemiae    hsemorrhagicae,   patho- 
genesis of,  432 

septicus  acuminatus,  502 

septicus  agrigenus,  442 

septicus  kerato-malaciae,  502 

septicus  sputigenus,  310 

septicus  ulceris  gangraenosi,  502 

septicus  vesicae,  505 

smaragdinus  foetidus,  453 

of  swine  plague,  Marseilles,  439 

of  symptomatic  anthrax,  542 

tenuis  sputigenus,  457 

tetani,  531 

tetani,  aerobic  cultures  of,  548 

tetani,  cultivation  of,  533 

tetani,  pathogenesis,  534 

of  Tornmasoli,  497 

of  Tricomi,  503 

tuberculosis,  393 

tuberculosis,  action  of  sunlight  on, 
403 

tuberculosis,    attenuation    of    viru- 
lence, 404 

tuberculosis,    biological    characters, 
397 

tuberculosis,    branching   forms   of, 
412 

tuberculosis,  in  bronchial  glands  of 
healthy  persons,  413 

tuberculosis,  cultivation  of,  399 
^tuberculosis,  duration    of    vitality, 
403 

tuberculosis,  morphology  of,  394 

tuberculosis,  pathogenesis,  408 

tuberculosis,    spore  formation   (?), 
398 

tuberculosis,  staining  of  the,  394 


Bacillus    tuberculosis,    thermal    death  - 
point,  154 

tuberculosis,  toxic  products  of,  405 

tuberculosis  galliriarum,  410 

typhi  abdominalis,  358 

typhi  abdominalis,  biological  char- 
acters,  359 

typhi     abdominalis,     detection     in 
water,  365-368 

typhi  abdominalis,  flagella  of,  359 

typhi  abdominalis,  morphology  of, 
358 

typhi  abdominalis,  pathogenesis,  364 

typhi  abdominalis,  pus  production 
by,  369 

typhi  abdominalis,    thermal  death- 
point,  363 

typhi    abdominalis,    toxic  products 
of,  363 

typhi  murium,  457 

typhosus,  358 

of  Unna  and  Hodara,  527 

of  Utpadel,  507 

varicosus  conjunctivae,  504 

venenosus,  513 

veneuosus  brevis,  513 

venenosus  invisibilis,  513 

venenosus  liquet'aciens,  514 
Bacteria  in  the  air,  methods  of  collect- 
ing, 625-631 

in  the  air,  results  of  researches,  632 

in  the  air,  in  school -rooms,  635 

in  the  air,  species  found,  634 

chemical  composition  of,  121 

of  mouth,  661.  666 

of  mouth,  peptonizing  action  of,  663 

in  the  soil,  652-657 

in  the  soil,  kinds  of,  654,  656 

in  the  soil,  method  of  studying,  652 

in  the  soil,  number  of,  653 

structure  of,  115 

on  surface  of  the  body,  658 

thermal  death -point  of,  150 

in  tissues,  methods  of  staining,  34 

in  water,  636-651 

in  water,  collection  of  water,  637 

in  water,  enumeration  of,  639,  641 

in  water,  species  found,  648 

in  wounds,  276 
Bacteridie  du  Charbon.  340 
Bacterie  septique,  583 
Bacteriology,  literature  of,  8 
Bacterium,  18 

ae"ruginosum,  479 

coli  commune,  462 

termo,  676 

Barium  chloride,  antiseptic  value  of,  184 
Bees,  infectious  disease  of,  507 
Beggiatoa,  19 

Benzene,  germicidal  value  of,  194 
Benzoic  acid,  germicidal  value  of,  179 
Benzo-naphthol,  germicidal  value  of,  202 
Beri-beri,  etiology  of,  573 


686 


INDEX. 


Binary  division,  117 

Biological  characters,  modifications  of, 

126-131 

Biskra  button,  etiology  of,  330 
Blood  serum,  as  a  culture  medium,  75 

serum,  coagulation  of,  56 

serum,  collection  of,  37 

serum,  germicidal  action  of,  204, 236 

serum,  sterilization  of,  55 
Booker's  bacilli,  468-472 
Boracic  acid,  germicidal  value  of,  178 
Bouillon,  41 
Bread,  bacteria  in,  682 
Brieger's  bacillus,  448 
Bromine,  germicidal  action  of,  174 
Bronchitis,  bacteria  in,  496,  574 
Broncho-pneumonia,  bacteria  in,  574 
Brownian  movement,  117 
Bubo,  bacteria  in,  574 
Bubonic  plague,  bacillus  of,  520 

plague  bacillus,  discovery  of,  8 
Bttffelseuche,  bacillus  of,  429 
Butter,  bacteria  of,  680 

"  cheesy,  "  bacteria  of,  680 

rancid,  bacteria  of,  680 
Butyric  acid,  germicidal  value  of,  179 

acid,  production  of,  134 

CADAVERIN,  143 
Cadavers,  bacteria  of,  674 
Calcium  chloride,  antiseptic  value  of, 
184 

hydroxide,  germicidal  value  of,  180 

hypochlorite,   germicidal   value  of, 
184 

light  for  photographing  bacteria,  106 
Camphor,  antiseptic  value  of,  194 
Capsule  bacilli,  517,  518,  519 

bacilli  in  ozana,  603 

composition  of,  116 
Carbol-fuchsin  solution,  29 
Carbolic  acid,  germicidal  value  of,  195 
Carbon,  how  obtained,  123 

dioxide,  not  a  germicide,  170 
Carbonic  oxide,  not  a  germicide,  171 
Carcinoma,  bacteria  in,  575 
Catarrh,  nasal,  294 
Catarrhal  inflammations,  226 
Caterpillars,  infectious  disease  of,  338 
Caucasian  milk  ferment,  136 
Cell  membrane,  115,  116 
Cerebro-spinal   meningitis,  etiology  of, 

575 

Cervix  uteri,  bacteria  in,  665 
Chalazion,  bacteria  in,  576 
Chancroid,  etiology  of,  57(1 
Charbon,  etiology  of,  339 

symptomatique,  bacillus  of,  542 
Cheese,  bacteria  in,  680 

ripening  of,  680 
Chemiotaxis,  247 

Chloral  hydrate,  antiseptic  value  of,  185 
Chlorine,  germicidal  action  of,  173 


Chloroform,  germicidal  action  of,  173 
Cholera,  Asiatic,  etiology  of,  578 

in  ducks,  bacillus  of,  434 

in  fowls,  bacillus  of,  429 

infantum,  bacteria  in,  468-471,  578 

infautum,  etiology  of,  487 

immunity,  568 

nostras,  bacteria  in,  561,  578 

des  poules,  bacillus  of,  429 

ptomaines,  146 

spirillum,  biological  characters,  553 

spirillum,  cholera-red  reaction,  557 

spirillum,    duration    of   vitality  in 
earth,  655 

spirillum,   duration    of    vitality    in 
water,  647 

spirillum,  morphological  characters, 
552 

spirillum,  pathogenesis,  560 

spirillum,  thermal  death-point,  556 

spirillum,  toxic  products  of,  558 

spirillum  in  water,  detection  of,  650 

in  swine,  bacillus  of,  434 
Cholin,  144 

Chromic  acid,  germicidal  action  of,  177 
Chromogenes,  14 
Chromogenic  bacteria,  132 

bacteria  in  milk,  678 
Chronic  infectious  diseases,  etiology  of, 

392 

Citric  acid,  germicidal  action  of,  178 
Cladpthrix,  19,  24 

Clarification  of  culture  media,  42,  43 
Classification,  10-19 

biological,  13 

morphological,  13 

of  Baumgarten,  12 

of  Colin,  11 

of  Davaine,  3,  10 

of  Dujardin,  3 

of  Ehrenberg,  3,  10 

of  Hoffmann,  10 

of  Nageli,  11 

of  Zopf,  12 
Clostridium,  18 
Coal-tar  products,  antiseptic  action  of, 

193-203 

Coffee  infusion,  germicidal  value  of,  196 
Cohn's  solution,  40,  123 
Cold,  germicidal  action  of,  149 
Colon  bacillus,  462 

bacillus  in  water,  detection  of,  650 
Colonies  of  bacteria,  70 

counting,  640 

Comma  bacillus  of  Koch,  552 
Conjunctiva,  bacteria  of,  659 
Conjunctivitis,  bacteria  in,  294,  507,  579 
Contact  preparations,  27 
"Corn-stalk  disease,  "  etiology  of,  580 
Cory/a,  bacteria  in,  581 
Cover-glass  preparations,  25 
Crenothrix,  19 
Creolin,  germicidal  value  of,  197 


INDEX. 


687 


Creosote,  germicidal  value  of,  197 
Cresol,  germicidal  value  of,  197 
Culture  media,  37-49 

media,  filtration  of,  42 

media,  liquid,  38-41 

media,  natural,  37 

media,  reaction  of,  124 

media,  solid,  41-49 
Cultures  in  liquid  media,  60-66 

on  potato,  76 

in  solid  media,  67-77 
Cultivation  of  anaerobic  bacteria,  78-85 
Cupric  chloride,  antiseptic  value  of,  185 

sulphate,  germicidal  value  of,  185 
Cystitis,  bacteria  in,  505,  581 

DARMBACILLUS  of  Schottelius,  497 
Decolorization,  28 
Defensive  proteids,  236 
Dengue,  bacteria  in,  584 
Dental  caries,  bacteria  in,  501,  584 
Desiccation,  germicidal  action  of,  155 
Diaphtherin,  germicidal  value  of,  198 
Diarrlioea,  bacteria  in,  585 

in  calves,  585 

green,  of  infants,  493 

summer,  etiology  of,  473 
Dimensions  of  bacteria,  20 
Dimethylamine,  144 
Diphtheria  bacillus,  antitoxin  of,  380 

antitoxin,  results  of  treatment,  386 

bacillus,  pseudo-,  380 

discovery  of,  7 

etiology  of,  371 

immunity,  379 

mixed  infection,  385 

toxin,  action  of,  225 

toxalbumin,  146 
Diphtheritic  inflammations,  225 
Diplococcus,  17 

intercellularis  meningitidis,  322 

of  pneumonia  in  horses,  334 

pneumoniae,  310 

Disinfecting  solutions,  standard,  208 
Disinfection  of  clothing,  bedding,  etc., 
209 

of  dead  bodies,  209 

in  diphtheria,  218 

of  excreta,  208,  213-217 

of  the  hands,  212 

of  merchandise  and  the  mails,  210 

practical  directions  for,  208-218 

of  privy  vaults,  217 

of  rags,  210 

of  railway  cars,  210 

of  ships,  210 

of  sick-room,  hospital  wards,  etc., 
209 

by  steam,  210 

Disinfektol,  germicidal  value  of,  198 
Distemper  in  dogs,  etiology  of,  586 
Distilled  water,  bacteria  in,  645 
Dogs,  infectious  diseases  of,  586 


Double  staining,  28 

Drop  cultures,  62 

Dust,  bacteria  in,  634,  635 

of  streets,  bacteria  in,  656 
Dysentery,  bacteria  in,  586 

ECLAMPSIA,  bacteria  in,  587 
Eczema,  bacteria  in,  587 

epizoOtica,  bacteria  in,  589 
Eggs  as  a  culture  medium,  40 

rotten,  bacteria  of,  682 
Egyptian  ophthalmia,  bacteria  in,  507, 

580,  599 

Ehrlich's  stain,  29 
Ehrlich-Weigert  method,  30 
Electric  light,  germicidal  action  of,  158 

light    for   photographing  bacteria, 

105 

Electricity,  germicidal  action  of,  158 
Eisner's  method,  367 
Emmerich's  bacillus,  462 
Empyema,  etiology  of,  589 
Endocarditis,  ulcerative,  283 

etiology  of,  590 
Endogenous  spores,  118 
Endometritis,  bacteria  in,  591 
Endosporium,  119 
Environment,  changes  due  to,  222 
Enzymes,  131 

Epidermis,  bacteria  of,  659 
Van  Ermengen's  method,  33 
Erlenmeyer  flasks,  61 
Erythema,  bacteria  in,  591 

nodosum,  bacillus  of  Demme,  494 
v.  Esmarch's  roll  tubes,  74 
Essential  oils,  antiseptic  value  of,  198 
Ether,  germicidal  value  of,  198 
Eucalyptol,  antiseptic  value  of,  199 
Euphorin,  antiseptic  value  of,  200 
Exospprium,  119 

Experiments  upon  animals,  94-100 
Eye,  inoculations  in,  97 

FACULTATIVE  anaerobics,  16 

parasites,  15,  125 
Faeces,  bacteria  of,  671 
Fermentation  tube,  66 
Ferric  chloride,  antiseptic  value  of,  185 
Ferrous  sulphate,  antiseptic  value  of,  185 
Filtration  of  culture  media,  42 
Finkler  and  Prior,  spirillum  of,  561 
Fiocca's  method,  32 

Fish,  infectious  diseases  of,  522,  523,  525 
"  Fixing  "  on  cover  glass,  26 
Flagella,  116 

methods  of  staining,  32 
Flesh-peptone-gelatin,  41,  67 

peptone  solution,  41 

Foot  and  mouth  disease,  bacteria  in,  589 
Formaldehyde,  germicidal  value  of,  200 
Formalin,  germicidal  value  of,  200 
Formic  acid,  germicidal  value  of,  179 
Foul  brood,  bacillus  of,  507 


f>88 


INDEX. 


Fowl  cholera,  bacillus  of,  429 
Freezing,  germicidal  action  of,  149 
Friedlander's  bacillus,  309 

method,  30 

Fungi,  spores  of,  in  the  air,  624 
Furunculosis,  etiology  of,  592 

GABBETT'S  method,  30 

Gallic  acid,  germicidal  value  of,  179 

Gangrene,  bacteria  in,  503,  592 

hospital,  224 

Gas  phlegmon,  etiology  of,  593 
Gases,  germicidal  action  of,  168-173 
Gaslight  for  photographing  bacteria,  107 
Gastric  juice,  germicidal  action  of,  668 
Gelatin,  liquefaction  of,  132 

nutrient,  preparation  of,  41 
Germicidal  action,  conditions  governing, 
165-167 

value,  tests  of,  163-165 
Glanders,  bacillus  of,  417 

bacillus,  discovery  of,  7 

diagnosis  of,  421 
Glycerin-agar,  43 

Gold  chloride,  germicidal  value  of,  186 
Gonococcus,  295 

discovery  of,  7 
Gram's  method,  29,  35 
Granuloma  fungoides,  593 
Green  pus,  bacillus  of,  479 
Growth,  conditions  of,  122-125 
Guaiacol,  germicidal  value  of,  200 

H.EMATOCOCCUS  bovis,  334 
Hasmoglobinuria  of  cattle,  etiology  of,  334 
Hail,  bacteria  in,  642 
Haloid   elements,  germicidal  action   of, 

173-175 
Heat,  dry,  action  of,  150 

germicidal  action  of,  149 

moist,  action  of,  150 
Heterogenesis,  5 
Hog  erysipelas,  bacillus  of,  442 
Holtz'  method,  365 
Hot  air  sterilizer,  52 
Hydrant  water,  bacteria  in,  644 
Ilydrocele  fluid,  as  a  culture  medium,  40 
Hydrochloric  acid,  germicidal  action  of, 

177 
Hydrofluoric  acid,  germicidal  action  of, 

175 
Hydrogen  apparatus,  83 

not  a  germicide,  169 

peroxide,  germicidal  action  of,  169 
Hydrophobia,  etiology  of,  593 

inoculations,  Pasteur's  method,  97, 

246 

Hydrosulphuric  acid,  germicidal  action 
of,  171 

acid,  production  of,  138 
Hydroxylamine,  germicidal  value  of,  200 

ICE,  bacteria  in,  643 


Ichthyol,  germicidal  value  of,  200 
Icterus,  infectious,  bacteria  in,  594 
Immunity,  acquired,  242 

acquired,  explanation  of,  274 

alkalinity  of  blood  a  factor,  238 

Buclmer's  experiments,  236,  237 

of  carnivora,  234 

due  to  antitoxins,  266 

exhaustion  theory,  249 

Hankin's  experiments,  237 

through  mother's  milk,  271 

neutralized  by  depressing  agencies, 
241 

neutralized  by  chemical  substances, 
240 

Pasteur's  method  of  producing,  245- 

race,  234 

relative  value  of,  241 

retention  theory,  251 

by  sterilized  cultures,  245 

from  vaccination,  244 

in  various  diseases  of  man,  243 

vital  resistance  theory,  252 
Impf tetanus  bacillus,  439 
Incubating  ovens,  86,  87 
Incubator  of  D' Arson val,  92 
Indol,  germicidal  value  of,  201 
Infection,  channels  of,  229-232 

general,  226 

influence  of  quantity,  227 

localized,  223 

mixed,  228 

rapidity  of,  230 

secondary,  227 

through  intestine,  230 

through  lungs,  230 

through  unbroken  skin,  229 

through  wounds,  229 
Infectious  diseases,  bacteria  in,  571-619 
Influenza  bacillus,  discovery  of,  8 

etiology  of,  387-391 

bacillus,  pseudo-,  391 

of  horses,  etiology  of,  334,  595 
Infusoria,  3 
Inoculation  experiments,    conditions  to 

be  observed,  98 

Inoculations,  technique  of,  95,  96 
Insects,  infectious  diseases  of,  595 
Intestine,  bacteria  of,  670,  673 
Involution  forms,  23 
Iodine,  germicidal  action  of,  173 

trichloride,  germicidal  action  of,  174 
lodoform,  germicidal  action  of,  174 

ether,  germicidal  action  of,  175 
lodol,  germicidal  action  of,  175 
Izal,  germicidal  value  of,  201 

JEQUIRITY  solution,  47 

KASESPIRILLEN,  563 
Herat itis,  etiology  of,  595 
Koch's  plate  method,  72 
syringe,  95 


INDEX. 


689 


Kruse's  method,  77 
Kiihne's  method,  35 

LACTIC  acid,  germicidal  action  of,  178 

acid,  production  of,  134 
Lake  water,  bacteria  in,  643 
Lanolin,  germicidal  value  of,  201 
Lead  chloride,  antiseptic  value  of,  186 

nitrate,  antiseptic  value  of,  186 
Leprosy  bacillus,  discovery  of,  7 

etiology  of,  414 
Leptothrix,  19 

Leucocytha?mia,  bacteria  in,  596 
Leuconostoc,  17 

Light,  germicidal  action  of,  155 
Liquefaction  of  gelatin,  132 
Liquefying      bacteria,      characters     of 

growth,  70 

Literature  of  bacteriology,  8 
Lithium    chloride,   antiseptic  value  of, 

186 

Lochial  discharge,  bacteria  of,  664 
Loffler's  method,  32 

solution,  29 

Loretin,  germicidal  value  of,  201 
Lymphangitis,  bacteria  in,  596 
Lysol,  germicidal  value  of,  201 

MADURA  foot,  bacteria  in,  596 
Malachite  green,   germicidal   value  of, 

194 

Malarial  diseases,  etiology  of,  596 
Malic  acid,  germicidal  value  of,  179 
Malignant  oedema,  537 

oedema,  immunity  from,  540 
Mallein,  148,  422 

"  Malta  fever, "  micrococci  in,  528,  597 
Manganese      protochloride,      antiseptic 

value  of,  186 

Marsh  gas,  production  of,  138 
Mastitis,  bacteria  in,  597 

bovine,  etiology  of,  329,  333,  338, 
528,  597 

in  sheep,  etiology  of,  332 
Measles,  bacteria  in,  515,  598 
Measurements  of  bacteria,  20 
Meat  infusions,  40 
Meats,  bacteria  in,  680 

smoked,  bacteria  in,  681 
Meatus  urinarius,  bacteria  of,  664,  666 
Meconium,  bacteria  in,  670 
Meningitis,  bacteria  in,  337,  504,  529 

Micrococcus    pneumonia?    crouposae 

in,  313 

Mercuric  chloride,  germicidal  value  of, 
186 

cyanide,  germicidal  value  of,  188 

iodide,  antiseptic  value  of,  188 
Mercury,  oxides  of,  antiseptic  value  of, 

189 

Merismopedia,  17,  22 
Metallic    salts,    germicidal    value    of, 
182-192 


Metchnikoff  theory,  256.  258-265 

Methane,  germicidal  action  of,  171 

Methylamin,  144 

Methyl -guanidin,  145 

Mice,  infectious  diseases  of,  598 

Micrococci,  general  characters  of,  17 

morphology  of,  21 
Micrococcus,  generic  characters,  17 
of  Almquist,  337 
askoformans,  327 
botryogenus,  327 
of  bovine  mastitis,  329 
of  bovine  pneumonia,  329 
of  Bruce,  528 
of  Demme,  331 
endocarditidis  rugatus,  332 
No.  II.  of  Fischel,  336 
of  Forbes,  338 
of   gangrenous  mastitis  in    sheep, 

332 

gingivae  pyogenes,  335 
gonorrhoea3,  295 
gonorrhceae,  cultivation  of,  297 
gonorrhoea?,  pathogenesis,  298 
of  Heydenreich,  330 
insectorum,  527 
of  Kirchner,  336 
lanceolatus,  310 
of  Manfredi,  328 
ovatus,  330 
Pasteuri,  310 
pneumonia?  crouposae,  310 
pneumonia?  crouposa?  in  abscesses, 

313 
pneumonia?     crouposa?,    action     of 

germicides  on,  316 
pneumonia?    crouposa?,     biological 

characters  of,  314 
pneumonia?  crouposa?  in  empyema, 

590 

pneumonia?  crouposae  in  endocardi- 
tis, 313,  590 
pneumonias    crouposa?   in    healthy 

eyes,  600 
pneumonia?     crouposa?,     immunity 

from,  316,  320 

pneumonia?    crouposa?    in    menin- 
gitis, 312 
pneumonia?  crouposae,  morphology 

of,  314 
pneumonia?      crouposa?      in     otitis 

media,  313,  601 
pneumonia?  crouposae,  pathogenesis, 

318 
pneumonia?  crouposae   in  pleuritis, 

606 
pneumonias  crouposse  in  pneumonic 

sputum,  311 
pneumonia?  crouposa?   in    purulent 

keratitis,  595 

pneumonia?  crouposa?  in  saliva,  310 
pneumonias  crouposae,  varieties  of, 

317 


690 


INDEX. 


Micrococcus  of  progressive  abscess  for- 
mation in  rabbits,  323 

of    progressive    tissue    necrosis    in 
mice,  323 

of  pyaemia  in  rabbits,  324 

pyogenes  tennis,  286 

salivarius  septicus,  324 

of  septicaemia  in  rabbits,  324 

subflavus,  324 

tetragenus,  326 

of  trachoma  ( ?) ,  325 
Micromillimetre,  20 
Microzyma  bombycis,  330 
Milk,  antitoxin  in,  270 

bacteria  in,  232,  677 

bitter,  bacteria  of,  680 

chromogenic  bacteria  in,  678 

coagulation  of,  677 

as  a  culture  medium,  39 

fermentation  of,  136,  677 

of  healthy  women,  bacteria  in,  679 

tubercle  bacillus  in,  679 

viscous  fermentation  of,  678 
Milzbrandbacillus,  340 
Modes  of  action,  221-228 
MOller's  method,  32 
Morphia  hydrochlorate,  antiseptic  value 

of,  189 

Morphology,  20-24 
Mould  fungi,  in  the  air,  624 
Mouse  septicaemia,  bacillus  of,  442 
Mouth,  bacteria  in,  661,  666 
Movements  due  to  flagella,  117 

character  of,  117 

Mucous  membranes,  bacteria  of,  659 
Mucus,  germicidal  action  of,  206 
Muscarin,  144 

Mustard,  oil  of,  antiseptic  value,  202 
Mykoprotein,  121 
Mytilotoxin,  145 

NAPHTHOL,  germicidal  value  of,  201 
Nasal  catarrh,  294 

mucus,  bacteria  in,  660 
Natural  immunity,  explanation  of,  239 
Neisser's  method,  31 
Nephritis,  bacteria  in,  495,  598 
Neuridin,  143 
Neurin,  144 

Nickel  sulphate,  antiseptic  value  of,  189 
Nitrates,  reduction  of,  140 
Nitric  acid,  germicidal  action  of,  177 
Nitrification,  140 
Nitrogen,  how  obtained,  123 

dioxide,  germicidal  action  of,  171 
Nitrous  acid,  germicidal  action  of,  177 

oxide,  not  a  germicide,  171 
Non- liquefy  ing  bacteria,    characters  of 

growth,  68 

Nose,  bacteria  in,  660,  666 
Nosema  bombycis,  330 
Nosophen,  germicidal  action  of,  175 
Nucleins,  germicidal  action  of,  206,  239 


Nucleus  in  bacteria,  115 

staining  of,  116 
Nutrient  agar,  43 

agar,  filtration  of,  44 

agar,  preparation  of,  46 

OIDIUM  lactis,  in  cheese,  680 

Oleic  acid,  germicidal  value  of,  179 

Ophthalmia,  etiology  of,  599 

Orthpchromatic  plates,  103 

Osmic  acid,  germicidal  action  of,  177 

Osteomyelitis,  282,  600 

Otitis  media,  Bacillus  pyocyaneus  in,  482 
media,  etiology  of,  293,  601 
media,      Micrococcus      pneumonia 
crouposae  in,  313 

Oxalic  acid,  germicidal  action  of,  178 

Oxygen,  germicidal  action  of,  168 
how  obtained,  122 

Ozaena,  bacteria  in,  496,  518,  602 

Ozone,  germicidal  action  of,  168 

PANARITIUM,  etiology  of,  603 
Panhistophytpn  ovatum,  330 
Panophthalmia,  bacteria  in,  600 
Papin's  digester,  54 
Parasites,  15,  221 

facultative,  124 

strict,  124 
Parasitism,  124 
Parietti's  method,  366 
Parotitis,  bacteria  in,  603 
Parrot  disease,  etiology  of,  338 
Pasteur-Chamberland  filter,  57 
Pasteur's  flasks,  61 

method  of  producing  immunity,  245 

solution,  40,  123 
Pathogenes,  14 
Pathogenic  bacteria,  15 

bacteria  in  water,  646 

bacteria  in  water,  detection  of,  650 

bacteria  in  water,  duration  of  vital- 
ity. 646 

power,  explanation  of,  222 

saprophytes,  462 
Pebrine,  etiology  of,  330 
Pemphigus,  bacteria  in,  331,  603 

neonatorum,  etiology  of,  337 
Peppermint,  oil  of,  antiseptic  value,  202 
Peptonizing  action  of  bacteria  in  saliva, 
663 

ferment,  132 
Peptotoxin,  145 
Pericarditis,  etiology  of,  604 
Periostitis,  etiology  of,  600 
Peritonitis,  etiology  of,  472,  604 
Petri's  dishes,  73 
Phagocytosis,  255 
Phenol,  germicidal  value  of,  195 
Phosphorescence,  141 
Phosphoric  acid,   germicidal  action  of, 

177 
Photographing  bacteria,  101-111 


INDEX. 


691 


Photographing  bacteria,    amplification, 

bacteria,  apparatus  required,  103 
Photomicrographs,  value  of,  101 
Photomicrography,    Borden's    method, 

109 

by  calcium  light,  106 
developing  solution,  111 
by  electric  light,  105 
by  gaslight,  107 
by  oil-light,  108 
Stern  berg's  method,  107 
by  sunlight,  105 
Phragmidiothrix,  19 
Phylaxins,  221 

Physical  agents,  influence  of,  149-159 
Pigment  production,  130 
Pittield's  method,  33 
Plants,  infectious  diseases  of,  605 
Plate  method  of  Koch,  639 
Platinum  bichloride,  antiseptic  value  of, 

189 

Pleuritis,  etiology  of,  606 
Pleuro-pneumonia    bacillus,    discovery 

of,  8 

of  cattle,  etiology  of,  499 
of  cattle,  protective  inoculations  in, 

500 

in  calves,  bacteria  in,  529 
Pneumobacillus  liquefaciens  bovis,  499 

septicus,  529 

Pneumococcus  (Friedlander) ,  308 
Pneumonia  antitoxin,  268 
bovine,  etiology  of,  329 
croupous,  etiology  of,  300-307,  607 
in  horses,  etiology  of,  334 
micrococcus,  discovery  of,  7 
Post-mortem  examinations,  99 
Potassium  acetate,  antiseptic  value  of, 

189 

arsenite,  antiseptic  value  of,  189 
bichromate,  antiseptic  value  of,  189 
bromide,  antiseptic  value  of,  189 
carbonate,  antiseptic  value  of,  189 
chlorate,  antiseptic  value  of,  189 
chromate,  antiseptic  value  of,  189 
cyanide,  antiseptic  value  of,  189 
hydroxide,  germicidal  value  of,  179 
iodide,  antiseptic  value  of,  189 
permanganate,  germicidal  value  of, 

190 

Potato  paste,  preparation  of,  48 
Pregl's  method,  36 
Pressure,  germicidal  action  of,  159 

regulator  of  Moitessier,  88 
Products  of  vital  activity,  130-142 
Proteus  capsulatus  septicus,  452 
fluorescens,  527 
Hauseri,  488 
of  Karlinsky,  489 
lethalis,  491 
mirabilis,  489 
septicus,  491 


Proteus  vulgaris,  485 

vulgaris  in  cholera  infantum,  487 

vulgaris  in  cystitis,  584 

vulgaris  in  pyelonephritis,  609 

Zenkeri,  491 
Protoplasm  of  bacteria,  116 

granules  in,  116 

Pseudo-diphtheritic  bacillus,  380 
Pseudo-diplococcus  pneumonias,  335 
Pseudo-leukaemia,  bacteria  in,  607 
Pseudo-tuberculosis,  bacteria  in,  608 
Ptomaines,  non -toxic,  143 

production  of,  222 

toxic,  144 

Puerperal  fever,  etiology  of,  608 
Pure  cultures,  37 

cultures  obtained  by  inoculation,  100 
Purpura  hrcmorrhagica,  bacteria  in,  510, 

511,  613 

Pus  cocci,  in  inflammations  of  mucous 
membranes,  293 

cocci  in  otitis  media,  293 

formation,  223,  275 
Putrefaction,  bacteria  of,  675 

products  of,  139 
Putrescin,  144 
PyaBmia,  definition  of,  608 
Pyelonephritis,  etiology  of,  608 
Pyocyanin,  131,  480 
Pyogenic  bacteria,  275-299 
Pyoktanin,  germicidal  value  of,  194 
Pyosalpinx,  bacteria  in,  610 

QUININE  hydrochlorate,  antiseptic  action 

of,  190 
sulphate,  antiseptic  value  of,  190 

RABBIT  septicaemia,  bacillus  of,  429 
Rain-water,  bacteria  in,  642 
Ranvier's  moist  chamber,  63 
Rauschbrand,  bacillus  of,  542 
Relapsing  fever,  etiology  of,  549 

fever  inoculation  experiments,  551 

fever  spirillum,  discovery  of,  7 
Reproduction,  by  binary  division,  117 

by  spores,  118 

rapidity  of,  118 

Rheumatic  fever,  etiology  of,  610 
Rhinitis  fibrinosa,  bacteria  in,  611 
Rhinoscleroma,  bacillus  of,  425 
Ricin,  experiments  of  Ehrlich,  270 
Rinderseuche,  bacillus  of,  429 
Roll  tubes,  v.  Esmarch's,  74 
Rothlauf,  bacillus  of,  442 
Rotz  bacillus,  417 
Rouget,  bacillus  of,  442 

protective  inoculations,  447 

SALICYLIC    acid,  germicidal    action  of, 

178 
Saliva,  bacteria  in,  662 

germicidal  action  of,  663 
Salomonson's  method,  77 


602 


INDEX. 


Salt,  not  a  germicide,  681 

Saprin,  144 

Saprol,  antiseptic  value  of,  202 

Saprophytes,  15 

Saprophytic  bacteria,  where  found,  623 

Sarcina,  17,  22 

Sarcinae,  from  stomach,  672 

Scarlet  fever,  bacteria  in,  612 

Scheinfaden,  118 

Schultz's  method,  46 

Schweineseuche,  bacillus  of,  429 

Scorbutus,  bacteria  in,  612 

Screens,   colored  in   photomicrography. 

103,  106 

Sea- water,  bacteria  in,  647,  651 
Sepsin  poisoning,  488 
Septicaemia,  226 

bacteria  in,  612 

in  cattle,  bacteria  in,  613 

definition  of,  428 

in  fowls,  bacteria  in,  613 

hnmorrhagica,  bacilli  in,  458 

in  swine,  bacteria  in,  613 
Sewers,  bacteria  in,  644 
Silicate  jelly,  47 
Silkworm,  diseases  of,  330 
Silver  chloride,  germicidal  value  of.  190 

nitrate,  germicidal  value  of,  190 
Skatol,  antiseptic  value  of,  202 
Small-pox,  antitoxin  of,  274 
Smear  preparations,  26 
Smoke,  antiseptic  value  of,  202 
Snake  venom,  antitoxin  of,  269 
Snow,  bacteria  in,  642 
Soap,  germicidal  value  of,  181 
Sodium  borate,  antiseptic  value  of,  191 

carbonate,  antiseptic  value  of,  191 

chloride,  antiseptic  value  of,  191 

hydroxide,  germicidal  value  of,  180 

hyposulphite,    antiseptic  value    of, 
191 

sulphite,  antiseptic  value  of,  191 
Solid  culture  media,  41 
Soluble  ferments,  140 
Sozoiodol    acid,   germicidal    action    of, 

175 

Spirilla,  morphology  of,  24 
Spirillum,  generic  characters,  18 

an  serum,  551 

cholera?  Asiatic*,  552-561 

choleras  Asiatics,  varieties  of,  565 

differential  diagnosis,  568 

of  Deneke,  563 

of  Finkler  and  Prior,  561 

Metchnikovi,  563 

Obermeieri,  549 

tyrogenum,  563 
Spirochaete,  18 

anserina,  551 

Obermeieri,  549 
Spirulina,  18 
Spontaneous  generation,  4 
Spores,  discovery  of,  5 


Spores,  formation  of.  118 

germination  of,  119 

location  of,  119 

methods  of  staining,  31 

thermal  death -point  of,  153 
Sputum,   examination    of,  for    tubercle 

bacilli,  395 
Stab  cultures,  87 
Staining  bacteria  to  photograph,  103 

bacteria  in  tissues,  34 

of  cover-glass  preparations,  27 

tlagella,  32 

methods,  25-36 

spores,  31 
Staphylococci,  21 

Staphylococcus  aureus,  toxic  products  of, 
147 

epidermidis  albus,  284 

pyogenes  albus,  284 

pyogenes  albus  in  "stitch  abscess, !r 
285 

pyogenes  aureus,  277-284 

pyogenes  aureus,  action  of  germici- 
dal agents,  278 

pyogenes  aureus,  pathogenesis,  280 

pyogenes   aureus,    pus    production 
by,  281 

pyogenes  citreus,  285 

pyosepticus,  337 

salivarius  pyogenes,  323 
Steam,  sterilization  by,  51 

sterilizers,  53 
Sterilization,  5 

of  culture  media,  50-59 

by  discontinuous  heating,  51 

by  dry  heat,  52 

by  moist  heat,  51 

by  filtration,  56 
Sterilizers,  hot  air,  52 

steam,  53 

Sternberg's  bulbs,  64 
Stick  cultures,  67 

cultures,  long,  for  anaCrobics,  7& 
Stomach,  bacteria  of,  668 

dilated,  bacteria  of,  672 
Stomatitis,  bacteria  in,  614 
Streak  cultures,  75 
Streptococci,  22 
Streptococcus,  generic  characters,  17 

agalactiau  contagiosae,  338 

articulorum,  290 

bombycis,  330 

of  Bonome,  337 

brevis,  287 

conglomeratus,  512 

coryza1  contagions  equorum,  334 

erysipelatos,  286 

lanceolatus  Pasteuri,  310 

longus,  287 

of  JVIunneberg,  331 

of  mastitis  in  cows,  333 

perniciosus  psittacorum,  338 

pyogenes,  286 


INDEX. 


693 


Streptococcus   pyogenes,  action  of  ger- 
micides upon,  289 

pyogenes,  in  diphtheria,  290 

pyogenes,  pathogenic  action  of,  289 

pyogenes,  in  puerperal  fever,  291 

pyogenes,    in  ulcerative  endocardi- 
tis, 290 

pyogenes  malignus,  292 

septicus,  330 

septicus  liquefaciens,  335 
Structure  of  bacteria,  115 
Sulphur  dioxide,  germicidal  action  of,  172 
Sulphuric  acid,  germicidal  action  of,  176 
Sulphurous  acid,  germicidal  action  of,  176 
Sunlight,  germicidal  action  of,  155 

for  photographing  bacteria,  105 
Susceptibility,  233 

of  young  animals,  233 
Swine  plague,  bacillus  of,  429 
Sycosis,  bacteria  in,  496 
Symbiosis,  125,  548 
Symptomatic  anthrax,  bacillus  of,  542 

immunity  from,  545 
Syphilis,  bacteria  in,  422-425 

TAXNIC  acid,  germicidal  value  of,  179 
Tartaric  acid,  germicidal  value  of,  179 
Temperature,  limits  of  growth,  123 
Tetanin.  146,  533 
Tetanotoxin,  146,  534 
Tetanus  antitoxin,  268,  537 

bacillus,  discovery  of,  8 

bacillus  of.  531-537 

bacillus,  toxic  products  of,  547 

immunitv,  536 
Tetrads,  22 

Texas  fever,  of  cattle,  etiology  of,  614 
Thermal  death-point  of  bacteria,  151 
Thermo-regulator  of  Bohr,  88 

of  Roux,  92-93 

of  Reichert,  88 
Thermo-regulators,  78-92 

electro-magnetic,  90,  93 
Thymic  acid,  germicidal  value  of,  179 
Thymol,  antiseptic  value  of,  203 
Thymus  bouillon,  246 
Tin  chloride,  antiseptic  value  of,  191 
Tobacco  smoke,  antiseptic  value  of,  203 
Torula  chains,  22 
Toxaemia,  definition  of,  428 
Toxalbumins,  146 

vegetable,  270 

Trachoma,  etiology  of,  294,  325,  614 
Trikresol,  germicidal  value  of,  198 
Trimethylamine,  144 
Tubercle  bacilli,  dead,  pathogenic  action 
of,  413 

bacilli  in  dust,  634,  635 

bacillus,    attenuation   of   virulence, 
413 

bacillus,    demonstration  of,  in  spu- 
tum. 414 

bacillus,  discovery  of,  392 


Tubercle  bacillus,  methods  of  staining,  30 

Tuberculin,  148 

Koch's  method  of  preparing,  406 
test  for  cattle,  407 

Tuberculosis,  acquired  immunity  from, 

407,  413 
in  cattle,  prevalence  of,  410 

Turpentine,  oil  of,  antiseptic  value,  202 

Typhoid  bacillus,  discovery  of,  7 

bacillus,    duration    of    vitality    in 

water,  647 

bacillus,  experiments  on  animals,  353 
bacillus,  toxic  products  of,  145,  147, 

356 

bacillus  in  water,  detection  of,  650 
bacillus,  where  found,  352 
fever,  etiology  of,  349-357 
infection,  predisposing  causes,  369 

Typhotoxin,  145 

Tyrotoxicon,  145,  678 

Typhus  fever,  bacteria  in,  555,  616 

ULCUS  corn*,  etiology  of,  595 
Underclothing,  bacteria  attached  to,  656 
Urea,  fermentation  of,  136 
Urine,  bacteria  in,  232 

as  a  culture  medium,  39 

germicidal  action  of,  207 
Urobacillus  liquefaciens  septieus,  582 

VACCINIA,  bacteria  in,  616 

Vagina,  bacteria  in,  664,  665 

Valerianic  acid,  germicidal  value  of,  179 

Varicella,  bacteria  in,  616 

Variola,  bacteria  in,  616 

Vegetable  infusions  as  culture  media,  41 

Vibrio,  18 

of  Asiatic  cholera,  552 

Metchnikovi,  563 

proteus,  561 
Vibrioniens,  3 
Vibrion  septique,  537 
Virulence,  attenuation  of,  127,  129 

recovery  of,  129 
Viscous  fermentation,  137 

fermentation  in  milk,  678 

WASH- WATER,  bacteria  in,  659 
Water,  pathogenic  bacteria  in,  646 
Wells,  bacteria  in,  644 
Whooping-cough,  bacteria  in,  617 
Wildseuche,  bacillus  of,  429 
Wool-sorter's  disease,  339 
Wurtz's  method,  367 

YELLOW-FEVER,  bacteria  in,  617 

ZIEIIL-NEELSON  method,  30,  35 
Ziehl's  solution,  29 
Zinc  chloride,  antiseptic  value  of,  191 
sulphate,  antiseptic  value  of,  192 
Zooglcea,  21 
Zymogenes,  14 


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RETURNED 

APR  14  1969 


CEIPI 


30m-10,'61  (C3941s4)4128 


QR46    Sternberg,  G.M. 

S83       Text-book  of  bacteriology 

1896 


6011)5 


